CN112980859B - Method for reducing stability of LKB1-STRAD-Mo25 protein complex - Google Patents
Method for reducing stability of LKB1-STRAD-Mo25 protein complex Download PDFInfo
- Publication number
- CN112980859B CN112980859B CN201911275064.1A CN201911275064A CN112980859B CN 112980859 B CN112980859 B CN 112980859B CN 201911275064 A CN201911275064 A CN 201911275064A CN 112980859 B CN112980859 B CN 112980859B
- Authority
- CN
- China
- Prior art keywords
- protein
- lkb1
- midkine
- strad
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for reducing the stability of an LKB1-STRAD-Mo25 protein complex. The stability of the LKB1-STRAD-Mo25 protein complex can be inhibited by increasing the expression level of the Midkine protein in human or mammalian cells. A Co-Immunoprecipitation (Co-IP) experiment is carried out by taking LKB1 as a bait protein, and a Western blot experiment is utilized to detect the protein levels of STRAD and Mo25 bound by LKB1, so that the negative regulation and control effect of the Midkine protein on the stability of an LKB1-STRAD-Mo25 protein complex can be verified. The invention provides a method for reducing the stability of the LKB1-STRAD-Mo25 protein complex, provides help for researching the biological function of the LKB1 protein, and provides a new theoretical basis for applying LKB1 as a target in tumors and diabetes.
Description
Technical Field
The invention belongs to the field of cell biology and transformation medicine, and particularly relates to a method for reducing the stability of LKB1-STRAD-Mo25 protein complex and identifying the complex.
Background
The Midkine protein is a secretable protein and after translation is complete, the Midkine molecule from which the signal peptide is removed is about 13 kD. Studies have shown that Midkine is widely expressed in human tissues and can be transported in vivo by secretion into the circulatory system. Midkine is widely involved in the regulation of various physiological processes such as immune inflammatory reaction, neural development, blood pressure regulation and the like. The latest clinical sample data show that the expression level of Midkine is in an up-regulation trend in various malignant tumors such as breast cancer, prostate cancer, liver cancer, pancreatic cancer and the like, and the survival rate of a patient group with high Midkine expression is remarkably reduced, which indicates that Midkine is a cancer promotion factor. Based on the above phenomena, the functional research of Midkine has been the focus of research.
According to reports, Midkine protein belongs to the Pleiotrophin (PTN) family. The classical theory is that Midkine protein secreted outside the cell influences the physiological behavior of the cell by binding with receptors on the cell membrane to regulate downstream signaling pathways. Cell membrane midkine receptors that have been found include alk (anaplastic lymphoma kinase), PTP ζ (protein tyrosine phosphatase ζ), Notch2, Low Density Lipoprotein (LDL) receptor LRP1(LDL receptor protein 1), and the like. Midkine can activate downstream signal pathways such as Akt and Erk and promote activities such as cell proliferation by binding with the receptors, but the mechanisms are not directly connected with the occurrence and development of tumors.
In previous studies, we found that Midkine protein has an inhibitory effect on the AMPK signaling pathway. AMPK (AMP-activated protein kinase) protein is a specific energy sensing and regulating factor of eukaryotes and is in central position in the regulation of metabolic balance of cells. AMPK is a heterotrimer composed of three subunits, α, β, γ, where β and γ are regulatory subunits and γ binds AMP (5' -adenosine monophosphate) to allosterically catalyze subunit α, activated by the upstream kinase LKB1 (lever kinase 1). When energy stress occurs and the ratio of AMP to ATP (5' -adenosine triphosphate) in cells changes, AMPK is greatly phosphorylated and activated, kinase activity is exerted, and a plurality of pathways such as glycolysis, fatty acid synthesis, beta oxidation, amino acid metabolism, glycogen synthesis, cell autophagy and the like are regulated and controlled by phosphorylating and activating or inhibiting a plurality of different downstream substrates, so that the purposes of promoting catabolism, inhibiting anabolism and stopping energy-consuming physiological processes are achieved, and the cells are adaptive to the environment of the energy stress. Midkine inhibits phosphorylation activation of AMPK. .
Meanwhile, we found that the inhibitory effect of Midkine on the AMPK pathway occurs inside cells by affecting LKB1 kinase activity. LKB1 belongs to the serine-threonine protein kinase family and can phosphorylate AMPK and its related family members. Unlike general protein kinases, LKB1 is not activated by phosphorylation by means of a Kinase or coupled receptor, but rather by forming a stable heterotrimer with the pseudokinase STRAD (STE20-Related Kinase adapter) and the scaffold protein Mo 25. The Mo25 protein provides a binding site for LKB1 and STRAD, so that the LKB1 and the STRAD can be tightly combined. The STRAD protein has a classical protein kinase domain, but several key amino acid residues are mutated and cannot bind ATP to function as a kinase. When LKB1-STRAD-Mo25 forms heterotrimers, spontaneous phosphorylation activation occurs on Ser and Thr residues at multiple sites such as 31, 189 and 325 on LKB1 protein, so that LKB1 has activity.
In the research, Midkine inhibits the formation of LKB1-STRAD-Mo25 protein complex, thereby inhibiting the activity of LKB1 and preventing the phosphorylation activation of AMPK by LKB 1.
LKB1 is an important cancer suppressor gene, and the downstream AMPK protein of LKB1 is closely related to tumors, diabetes mellitus and the like. The Midkine is found to be used for regulating the stability of the LKB1-STRAD-Mo25 protein complex, so that the Midkine-LKB1-AMPK pathway action mechanism is further disclosed, and the Midkine-LKB1-AMPK pathway protein complex is used for clinical transformation application and has an important theoretical support effect.
Disclosure of Invention
1. The invention aims to provide a method for reducing the stability of LKB1-STRAD-Mo25 protein complex, which can be used for researching LKB 1-related signal channels and providing theoretical basis for taking LKB1 as a clinical treatment target.
2. The low-activity LKB1 protein provided by the invention is realized by preventing LKB1 from forming a complex with STRAD and Mo25 protein through high expression of Midkine protein in cells, so that the self-activation of LKB1 is inhibited, the regulation and control of LKB1 protein kinase activity are realized in vivo on the premise of no need of gene knockout operation, and conditions are provided for researching functions of proteins such as LKB1 and downstream AMPK.
3. A method for reducing the stability of LKB1-STRAD-Mo25 protein complex, comprising the following steps:
(1) culturing cells expressing LKB 1;
(2) the cells were treated according to 2-5X106Cell/dish number cells were seeded into 10cm culture dishes and cultured in a medium containing 10% fetal bovine serum (FBS, sera, S-FBS-SA-015);
(3) after 24 hours, after the cells adhere to the wall, the culture medium is replaced, and fresh culture medium containing 10% fetal bovine serum (FBS, SERANA, S-FBS-SA-015) is used for culture;
(4) preparing a Transfection system, adding 2-5 μ g of pCDNA3.1 and pCDNA-MDK plasmid into 250 μ l of Opti-MEM culture medium (gibco, 31985), mixing, taking 2 parts of TurboFect Transfection Reagent (Thermo, R0531)12 μ l, mixing with 250 μ l of Opti-MEM culture medium (gibco, 31985), mixing the Opti-MEM containing plasmid and TurboFect Transfection Reagent, and standing at room temperature for 30 min;
(5) adding the transfection system into a cell culture medium, and continuously culturing for 36-72 hours;
(6) after continuing culturing for 36-72 hours, sucking away the culture medium of the transfected cells, adding 1-2ml of PBS (Hyclone, SH30256.01) into each culture dish to wash the residual culture medium, sucking away the PBS, and putting the cell culture dish into liquid nitrogen for quenching;
(7) preparing NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40), and adding protease inhibitor (Selleck, B14002) and phosphatase inhibitor (Selleck, B15002) for standby;
(8) 1ml of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) was added per 10cm dish, the cells were scraped off with a cell scraper (Corning, 3008) and transferred to a pre-cooled 1.5ml centrifuge tube;
(9) rotationally incubating the cell lysate for 1-4 hours at 4 ℃ to fully lyse the cells;
(10) centrifuging at 4 deg.C and 12000rpm for 15-30min, and collecting supernatant to obtain cell lysate;
(11) obtaining a cell protein sample by taking 20-100 mul from the lysate, adding 4X Loading Buffer (Takara, 9173) according to the proportion, denaturing at 97 ℃ for 10 minutes to be used as an input sample, and storing at-30 ℃ or directly preparing for electrophoretic detection;
(12) adding 5-30 μ l of beads (agarose beads) capable of binding to LKB1 protein to the rest of the protein lysate, and performing rotary incubation at 4 ℃ for 1-6 hours or overnight incubation, so that LKB1 protein and proteins mutually binding to LKB1, such as STRAD, Mo25 and the like, are bound to the beads (agarose beads);
(13) centrifuging at 4 ℃ for 3-5 minutes by using a centrifugal force of 600-;
(14) centrifuging for 3-5 minutes at 4 ℃ by using a centrifugal force of 600-1000g, sucking away the supernatant, and repeatedly rinsing for 3-5 times;
(15) after the last rinse, the supernatant was aspirated, 50-150. mu.l of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) containing inhibitors (Selleck, B14002, B15002) were added and 4X Loading Buffer (Takara, 9173) was added in proportion, denatured at 97 ℃ for 10min, used as an IP sample, stored at-30 ℃ or directly prepared for electrophoretic detection;
(16) adding the denatured protein sample into the sample loading well, performing SDS-PAGE electrophoresis, transferring the protein on the acrylamide gel to 0.45 μm PVDF membrane by wet transfer after electrophoresis, incubating for 1-3 hours with 5% skimmed milk powder (BD, 232100) for blocking, incubating for blocking with beta-actin (protein, 20536-1-AP) and midkine (Origene, TA310977), AMPK alpha (CST, 5831), STRAD (Abcam, ab192879), Mo25(Abcam, ab51132), LKB1(CST, 3047) specific rabbit-derived primary antibody for overnight at 4 deg.C, recovering the primary antibody on the second day, washing the PVDF membrane three times with TBST buffer (10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20), adding 10min each time, adding goat anti-rabbit (AP, AP-1 mM) dissolved in 5% skimmed milk powder (BD, 232100), reacting for 10min at room temperature, 150mM NaCl, 0.1% Tween-20) washing the PVDF membrane for three times, 10min each time, and finishing the light-emitting preparation work;
(17) ECL luminescence solution (Tanon, 180-501) is used for carrying out chemiluminescence reaction, and the protein expression level is detected. A significant reduction in the number of STRAD and Mo25 proteins bound by LKB1 was detected in Midkine-highly expressing cells compared to controls;
the invention discloses a method for reducing the stability of an LKB1-STRAD-Mo25 protein complex. The Midkine protein is highly expressed or not expressed in the cells, Co-Immunoprecipitation (Co-IP) experiments are carried out by taking LKB1 as a decoy protein, protein levels of STRAD and Mo25 bound to LKB1 are detected by Western blot experiments, and it is found that the ability of the LKB1 protein to bind to the STRAD and Mo25 proteins is significantly reduced in the Midkine-highly expressed cells compared with the cells not expressing the Midkine protein. The results prove that the Midkine protein can inhibit the formation of LKB1-STRAD-Mo25 protein complex. Because the LKB1 protein is mainly self-activated by forming a complex with the STRAD and the Mo25 protein to obtain kinase activity, the establishment of the method provides a method for reducing the stability of the LKB1-STRAD-Mo25 protein complex, provides help for researching the biological function of the LKB1 protein, and provides a new theoretical basis for applying LKB1 as a target in tumors and diabetes.
Compared with the prior art, the application of the invention utilizes the new function of the Midkine protein, reduces the binding capacity of the LKB1 protein with the STRAD and Mo25 protein without gene knockout and other operations, inhibits the self-activation of LKB1 and the kinase activity thereof in an in vivo environment, and creates conditions for researching the functions of LKB1 and downstream substrate proteins thereof and the application of the LKB1 and the downstream substrate proteins as clinical treatment targets.
Drawings
FIG. 1: the stability of the LKB1-STRAD-Mo25 protein complex is reduced by high expression of Midkine. (A) Expressing fusion Flag-tagged LKB1 protein in 293T cells, and detecting the effect of Midkine on the protein levels of STRAD and Mo25 bound by LKB1 through a WB experiment; (B) the SFB-tagged LKB1 fusion proteins were expressed in 293T cells and the effect of different Midkine expression levels in the cells on LKB 1-bound STRAD and Mo25 protein levels was examined by WB experiments.
Detailed Description
The effect of Midkine on the stability of the LKB1-STRAD-Mo25 protein complex was examined in the following manner by transiently expressing LKB1 and Midkine proteins in 293T cells as an example, and the effect of the present invention is described in detail
Example 1. detection of the effect of Midkine on the binding of Flag-LKB1 protein to STRAD and Mo25 protein in 293T cells.
(1) Culturing expression 293T cells;
(2) 293T cells according to 2.5X106Cell/dish number cells were seeded into 10cm dishes and plated with a solution containing 10% fetal bovine serum (FBS, SERANA, S-FBS-SA-015)Culturing the culture medium;
(3) after 24 hours, after the cells adhere to the wall, the culture medium is replaced, and fresh culture medium containing 10% fetal bovine serum (FBS, SERANA, S-FBS-SA-015) is used for culture;
(4) preparing a Transfection system, mixing 5 mu g of pCDNA3.1 and pCDNA-MDK plasmids with 3 mu g of Flag-LKB1 respectively, adding the mixture into 250 mu l of Opti-MEM culture medium (gibco, 31985), uniformly mixing, taking 12 mu l of TurboFect Transfection Reagent (Thermo, R0531) respectively, mixing with 250 mu l of Opti-MEM culture medium (gibco, 31985), mixing the Opti-MEM containing the plasmids and the TurboFect Transfection Reagent respectively, and standing at room temperature for 30 min;
(5) adding 2 parts of transfection system into 1 293T cell culture medium, and continuously culturing for 48 hours;
(6) the medium of the transfected cells was aspirated, 2ml of PBS (Hyclone, SH30256.01) was added to each dish to wash the remaining medium, the PBS was aspirated, and the cell culture dishes were quenched in liquid nitrogen;
(7) preparing NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40), and adding protease inhibitor (Selleck, B14002) and phosphatase inhibitor (Selleck, B15002) for standby;
(8) 1ml of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) was added per 10cm dish, the cells were scraped off with a cell scraper (Corning, 3008) and transferred to a pre-cooled 1.5ml centrifuge tube;
(9) rotationally incubating the cell lysate for 2 hours at 4 ℃ to fully lyse the cells;
(10) centrifuging at 4 deg.C and 12000rpm for 15min, and collecting supernatant to obtain cell lysate;
(11) obtaining a cell protein sample by taking 100 mul of lysate, adding 4X Loading Buffer (Takara, 9173) according to a proportion, denaturing at 97 ℃ for 10 minutes to be used as an input sample, and storing at-30 ℃ or directly preparing for electrophoretic detection;
(12) adding 20. mu.l Protein A/G Plus Agarose (Pierce, 20423) to the remaining Protein lysate, incubating at room temperature for 2 hours, adding 5. mu.g Normal Rabbit IgG (CST, 2729), continuing incubation at room temperature for 2 hours, and removing non-specifically bound Protein;
(13) using 900g of centrifugal force, 4 degrees C centrifugal 5 minutes, suction supernatant, adding 800L containing inhibitors (Selleck, B14002, B15002) NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40)4 degrees C spin incubation for 10 minutes, the beads (agarose beads) are rinsed;
(14) centrifuging at 4 deg.C for 4 min with 900g centrifugal force, sucking supernatant, and repeatedly rinsing for 4 times;
(15) after the last rinse, the supernatant was transferred to a new 1.5ml centrifuge tube, 100. mu.l of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) containing the inhibitor (Selleck, B14002, B15002) was added and 4X Loading Buffer (Takara, 9173) was added in proportion, denatured at 97 ℃ for 10min, as an IgG sample, stored at-30 ℃ or directly prepared for electrophoretic detection;
(16) add 20. mu.l Protein A/G Plus Agarose (Pierce, 20423) and 10LKB1 specific antibody (CST, 3047) to the Protein supernatant samples and incubate overnight at 4 ℃;
(17) centrifuging at 4 ℃ for 3-5 minutes by using the centrifugal force of 600-1000g, sucking the supernatant, adding 800. mu.l of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) containing an inhibitor (Selleck, B14002, B15002) and performing rotary incubation at 4 ℃ for 5-15 minutes to rinse the beads (agarose beads);
(18) after the last rinse, the supernatant was aspirated, 100. mu.l of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) containing inhibitors (Selleck, B14002, B15002) were added and 4X Loading Buffer (Takara, 9173) was added in proportion, denatured at 97 ℃ for 10 minutes, used as an IP sample, stored at-30 ℃ or directly prepared for electrophoretic detection;
(16) the denatured Protein samples were applied to the wells for SDS-PAGE, after which the proteins on the acrylamide gel were transferred to 0.45 μm PVDF membrane by wet transfer, blocked by incubation with 5% skim milk (BD, 232100) for 1-3 hours, and incubated overnight at 4 ℃ with midkine (Origene, TA310977), STRAD (Abcam, ab192879), Mo25(Abcam, ab51132), Flag (Protein, 66008-2-Ig) specific rabbit origin primary antibody, respectively. The next day, primary antibody was recovered, PVDF membrane was washed with TBST buffer (10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20) three times for 10min each, goat anti-rabbit secondary antibody (Millipore, AP-132-P) dissolved in 5% skim milk powder (BD, 232100) was added, reaction was carried out at room temperature for 1 hour, PVDF membrane was washed with TBST buffer (10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20) three times for 10min each, and light emission preparation work was completed;
(17) ECL luminescence solution (Tanon, 180-501) is used for carrying out chemiluminescence reaction, and the protein expression level is detected. A significant reduction in the levels of STRAD and Mo25 protein bound by LKB1 protein (represented by a Flag tag) was detectable in Midkine protein expressing cells compared to controls (fig. 1A). Example 2 Midkine protein was expressed at different concentrations in 293T cells and tested for its effect on the binding of SFB-LKB1 fusion protein to STRAD and Mo25 proteins.
(1) Culturing expression 293T cells;
(2) 293T cells according to 2.5X106Cell/dish number cells were seeded into 10cm culture dishes and cultured in a medium containing 10% fetal bovine serum (FBS, sera, S-FBS-SA-015);
(3) after 24 hours, after the cells adhere to the wall, the culture medium is replaced, and fresh culture medium containing 10% fetal bovine serum (FBS, SERANA, S-FBS-SA-015) is used for culture;
(4) preparing a Transfection system, mixing 0, 2, 4 and 6 mu g of pCDNA-MDK plasmid with 3 mu g of SFB-LKB1 (instantly expressing LKB1 protein fused with Falg and S-protein labels), supplementing 9 mu g of plasmid, adding the plasmid into 250 mu l of Opti-MEM culture medium (gibco, 31985), mixing uniformly, mixing 2 parts of TurboFect Transfection Reagent (Thermo, R0531) with 12 mu l of Opti-MEM culture medium (gibco, 31985), mixing the plasmid with the Opti-MEM containing TurboFeTransfection Reagent, and standing at room temperature for 30 min;
(5) adding 4 parts of transfection systems into 1 293T cell culture medium respectively, and continuously culturing for 48 hours;
(6) the medium of the transfected cells was aspirated, 2ml of PBS (Hyclone, SH30256.01) was added to each dish to wash the remaining medium, the PBS was aspirated, and the cell culture dishes were quenched in liquid nitrogen;
(7) preparing NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40), and adding protease inhibitor (Selleck, B14002) and phosphatase inhibitor (Selleck, B15002) for standby;
(8) 1ml of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) was added per 10cm dish, the cells were scraped off with a cell scraper (Corning, 3008) and transferred to a pre-cooled 1.5ml centrifuge tube;
(9) rotationally incubating the cell lysate for 2 hours at 4 ℃ to fully lyse the cells;
(10) centrifuging at 4 deg.C and 12000rpm for 15min, and collecting supernatant to obtain cell lysate;
(11) obtaining a cell protein sample by taking 100 mul of lysate, adding 4X Loading Buffer (Takara, 9173) according to a proportion, denaturing at 97 ℃ for 10 minutes to be used as an input sample, and storing at-30 ℃ or directly preparing for electrophoretic detection;
(12) adding 25 μ l S-Protein Agarose (Novagen, 69702) to the remaining Protein lysate, and rotary incubating at 4 deg.C for 4 hours to allow LKB1 Protein and proteins that bind to LKB1, such as STRAD, Mo25, etc., to bind to beads;
(13) using 900g centrifugal force, 4 degrees C centrifugal 5 minutes, suction supernatant, adding 750L containing inhibitors (Selleck, B14002, B15002) NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40)4 degrees C spin incubation for 10 minutes, the beads (agarose beads) for rinsing;
(14) centrifuging for 5 minutes at 4 ℃ by using 900g of centrifugal force, sucking away supernatant, and repeatedly rinsing for 4 times;
(15) after the last rinse, the supernatant was aspirated, 100. mu.l of NETN lysate (20mM Tris-base, 100mM NaCl, 1mM EDTA, 0.5% Nonidet p-40) containing inhibitors (Selleck, B14002, B15002) were added, and 4X Loading Buffer (Takara, 9173) was added in proportion, denatured at 97 ℃ for 10 minutes, as an IP sample, stored at-30 ℃ or directly prepared for electrophoretic detection;
(16) adding the denatured protein sample into the sample loading well, performing SDS-PAGE electrophoresis, transferring the protein on the acrylamide gel to 0.45 μm PVDF membrane by wet transfer after electrophoresis, incubating for 1-3 hours with 5% skimmed milk powder (BD, 232100) for blocking, incubating for overnight with Midkine (Origene, TA310977), AMPK α (CST, 5831), STRAD (Abcam, ab192879), Mo25(Abcam, ab51132), LKB1 (CSTR, 3047) specific rabbit primary antibody at 4 ℃ overnight, collecting the primary antibody the next day, washing the PVDF membrane three times with TBST buffer (10mM Tris-HCl, 150mM NaCl, 0.1% Tween-20), washing the PVDF membrane 10min each time, adding sheep anti-rabbit secondary antibody (NaCl, AP-132-P) dissolved in 5% milk powder (BD, Tris 100), reacting for 1 hour, washing the PVDF membrane three times with room temperature buffer (TBST-150 mM) and 150mM NaCl, 150% Tween-20, finishing the light-emitting preparation work every 10 min;
(17) ECL luminescence solution (Tanon, 180-501) is used for carrying out chemiluminescence reaction, and the protein expression level is detected. It can be detected that with the increase of the amount of transfected Midkine expression plasmid, the expression level of Midkine in the cells is gradually increased, while the levels of STRAD and Mo25 bound by LKB1 are gradually decreased, and the ability of LKB1 to bind to its substrate AMPK α is not significantly changed, which proves that Midkine specifically reduces the stability of the LKB1-STRAD-Mo25 protein complex.
Claims (2)
1. A method for reducing the stability of LKB1-STRAD-Mo25 protein complexes,
the expression plasmid with Midkine coding gene is transfected in the isolated cells of human cells or mammalian cells to improve the expression quantity of Midkine protein.
2. The method of claim 1,
the confirmation process of reducing the stability of LKB1-STRAD-Mo25 protein complex by increasing Midkine protein expression level:
1) culturing in vitro cells of human cells or mammalian cells, transfecting the cells with expression plasmids with Midkine coding genes, and performing high expression on Midkine proteins; meanwhile, the same cell is transfected with an expression plasmid without Midkine coding gene, and the expression plasmid is used as a control group cell without high expression Midkine protein;
2) for cells with high expression of Midkine and cells of a control group thereof, an LKB1 is used as a bait protein to carry out an immune co-precipitation experiment, a Western blot experiment is used for detecting the capacity of the STRAD and the Mo25 proteins combined with LKB1, and the finding that the capacity of the LKB1 protein combined with the STRAD and the Mo25 protein is obviously reduced in the cells with high expression of Midkine compared with the cells without expression of Midkine protein proves that the Midkine protein can inhibit the formation of an LKB1-STRAD-Mo25 protein complex.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911275064.1A CN112980859B (en) | 2019-12-12 | 2019-12-12 | Method for reducing stability of LKB1-STRAD-Mo25 protein complex |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911275064.1A CN112980859B (en) | 2019-12-12 | 2019-12-12 | Method for reducing stability of LKB1-STRAD-Mo25 protein complex |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112980859A CN112980859A (en) | 2021-06-18 |
| CN112980859B true CN112980859B (en) | 2022-07-05 |
Family
ID=76331591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911275064.1A Active CN112980859B (en) | 2019-12-12 | 2019-12-12 | Method for reducing stability of LKB1-STRAD-Mo25 protein complex |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112980859B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913421A (en) * | 2017-12-12 | 2019-06-21 | 中国科学院大连化学物理研究所 | The building of the protein stabilized high expression tumour cell system of midkine and identification method |
| CN111254175A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院大连化学物理研究所 | Method for collecting Midkine protein in vitro and for treating cells |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2402938B (en) * | 2003-06-17 | 2005-11-09 | Medical Res Council | Kinase assay |
| WO2005010174A2 (en) * | 2003-07-17 | 2005-02-03 | University Of Dundee | Methods for use of an lkb1/strad7mo25 complex |
-
2019
- 2019-12-12 CN CN201911275064.1A patent/CN112980859B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913421A (en) * | 2017-12-12 | 2019-06-21 | 中国科学院大连化学物理研究所 | The building of the protein stabilized high expression tumour cell system of midkine and identification method |
| CN111254175A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院大连化学物理研究所 | Method for collecting Midkine protein in vitro and for treating cells |
Non-Patent Citations (4)
| Title |
|---|
| LKB1抑制肿瘤机制的研究进展;杨事达等;《国际检验医学杂志》;20130630;第34卷(第12期);1557-1559 * |
| Midkine noncanonically suppresses AMPK activation through disrupting the LKB1-STRAD-Mo25 complex;Tian Xia等;《bioRxiv preprint》;20210928;全文 * |
| 应用酵母双杂交系统筛选LKBl相互作用蛋白;徐卿等;《安徽医科大学学报》;20180211;第53卷(第2期);第232-235页 * |
| 植物多酚通过MAPK信号通路调控肿瘤作用机制研究进展;李瑶等;《食品科学》;20171231;第38卷(第07期);296-301 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112980859A (en) | 2021-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Martin et al. | The mTOR/p70 S6K1 pathway regulates vascular smooth muscle cell differentiation | |
| Sato et al. | A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction | |
| Liu et al. | Metastatic breast cancer cells overexpress and secrete miR-218 to regulate type I collagen deposition by osteoblasts | |
| Rangaswami et al. | Cyclic GMP and protein kinase G control a Src-containing mechanosome in osteoblasts | |
| Lin et al. | LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration | |
| Chen et al. | Vgl-4, a novel member of the vestigial-like family of transcription cofactors, regulates α1-adrenergic activation of gene expression in cardiac myocytes | |
| Rajasingh et al. | Cell-free embryonic stem cell extract–mediated derivation of multipotent stem cells from NIH3T3 fibroblasts for functional and anatomical ischemic tissue repair | |
| Csukasi et al. | The PTH/PTHrP-SIK3 pathway affects skeletogenesis through altered mTOR signaling | |
| Kwak et al. | Nuclear accumulation of globular actin as a cellular senescence marker | |
| Dey et al. | Cyclic GMP–Dependent Protein Kinase Inhibits Osteopontin and Thrombospondin Production in Rat Aortic Smooth Muscle Cells | |
| Ryu et al. | Autotaxin-LPA axis regulates hMSC migration by adherent junction disruption and cytoskeletal rearrangement via LPAR1/3-dependent PKC/GSK3β/β-catenin and PKC/Rho GTPase pathways | |
| Borges et al. | Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells | |
| Zhen et al. | miR-301a-PTEN-AKT signaling induces cardiomyocyte proliferation and promotes cardiac repair post-MI | |
| Ambesi et al. | Anastellin, a fragment of the first type III repeat of fibronectin, inhibits extracellular signal-regulated kinase and causes G1 arrest in human microvessel endothelial cells | |
| Fukushima et al. | Loss of ΔNp63α promotes invasion of urothelial carcinomas via N-cadherin/Src homology and collagen/extracellular signal-regulated kinase pathway | |
| Arvisais et al. | AKT-independent phosphorylation of TSC2 and activation of mTOR and ribosomal protein S6 kinase signaling by prostaglandin F2α | |
| Chaterji et al. | Syndecan-1 regulates vascular smooth muscle cell phenotype | |
| Li et al. | Clk/STY (cdc2-like kinase 1) and Akt regulate alternative splicing and adipogenesis in 3T3-L1 pre-adipocytes | |
| Jin et al. | RNA interference elucidates the role of focal adhesion kinase in HLA class I-mediated focal adhesion complex formation and proliferation in human endothelial cells | |
| Wieland | Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability | |
| Yang et al. | Mechanisms by which the inhibition of specific intracellular signaling pathways increase osteoblast proliferation on apatite surfaces | |
| Sun et al. | Transcribed ultraconserved regions, Uc. 323, ameliorates cardiac hypertrophy by regulating the transcription of CPT1b (carnitine palmitoyl transferase 1b) | |
| WO2019157495A2 (en) | Methods for preventing and/or treating bone loss conditions by modulating irisin | |
| Yeo et al. | Src SH2 arginine 175 is required for cell motility: specific focal adhesion kinase targeting and focal adhesion assembly function | |
| Hayes et al. | Ttc39c is upregulated during skeletal muscle atrophy and modulates ERK1/2 MAP kinase and hedgehog signaling |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |