Anti gsk3b

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-GSK3β is a primary antibody that specifically recognizes glycogen synthase kinase 3 beta (GSK3β), a serine/threonine protein kinase involved in the regulation of various cellular processes. This antibody can be used for the detection and analysis of GSK3β in a range of applications, including Western blotting, immunoprecipitation, and immunohistochemistry.

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Anti-phospho-GSK3b GSK3B

7 protocols using anti gsk3b

Western Blot Analysis of Signaling Pathways

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The cells were lysed using mammalian protein extraction reagent RIPA (Beyotime, Haimen, China) supplemented with protease inhibitors cocktail (Roche) and PMSF (Roche). Fifty micrograms of the protein extractions were separated by 10% SDS-PAGE transferred to 0.22 mm nitrocellulose (NC) membranes (Sigma-Aldrich) and incubated with specific antibodies.The autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, CA, USA). Anti-EZH2 was from Abcam (Hong Kong, China). Anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-phospho-GSK3b and anti-GSK3b were from Cell Signaling Technology (Boston, MA, USA). Anti-HOXB7 was from Abcam. Anti-p53 was from Santa Cruz Biotechnology (Dallas, TX, USA). Results were normalized to the expression of GAPDH (Rabbit anti-GAPDH).

Zhang E.B., Yin D.D., Sun M., Kong R., Liu X.H., You L.H., Han L., Xia R., Wang K.M., Yang J.S., De W., Shu Y.Q, & Wang Z.X. (2014). P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression. Cell Death & Disease, 5(5), e1243-.

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Western Blot Antibody Information

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Western blot was performed as described.^{(32 (link))} Antibody information is described as follows. Anti-TSC1: #4906, Cell Signaling Technology, Beverly, MA, USA; anti-β-catenin: #8480, Cell Signaling Technology, Beverly, MA, USA; anti-active β-catenin: #8814, Cell Signaling Technology, Beverly, MA, USA; anti-p-GSK-3b (S9A): #5558, Cell Signaling Technology, Beverly, MA, USA; anti-GSK-3b: #9315, Cell Signaling Technology, Beverly, MA, USA; anti-p-S6: #5364, Cell Signaling Technology, Beverly, MA, USA; anti-S6: #2217, Cell Signaling Technology, Beverly, MA, USA; anti-p62: #5114, Cell Signaling Technology, Beverly, MA, USA; anti-LC3: #2775, Cell Signaling Technology, Beverly, MA, USA; anti-Vinculin: #V4505, Sigma-Aldrich, St. Louis, MO, USA; anti-a-Tubulin: #T6199, Sigma-Aldrich, St. Louis, MO, USA; and anti-Notch1: #ab52627; Abcam, Cambridge, MA, USA.

Choi H.K., Yuan H., Fang F., Wei X., Liu L., Li Q., Guan J.L, & Liu F. (2018). Tsc1 Regulates the Balance Between Osteoblast and Adipocyte Differentiation Through Autophagy/Notch1/β-Catenin Cascade. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 33(11), 2021-2034.

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Western Blot and Immunofluorescence Analysis

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Cells were washed with phosphate buffered solution (PBS) and whole cell lysate was prepared with modified RIPA buffer. Supernatants of the homogenates were subjected to 4%–12% Bis-Tris gel by electrophoresis, and transferred to PVDF membranes. The membranes were probed with anti-vimentin, anti-GAPDH, anti-rabbit or mouse IgG horseradish peroxidase (Sigma, St. Louis, MO, USA), anti-BMI-1 (Millipore Inc., Billerica, MA, USA), anti-AKT, anti-p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GSK3b, anti-p-GSK3b, anti-snail, anti-α-catenin, anti-β-catenin (Cell Signaling Technology, Beverly, CA, USA) and detected with ECL Western blotting detection reagents (Thermo Fisher, Palo Alto, CA, USA).
Immunofluorescence staining was performed on cells plated in chamber slides. The cells were fixed in 3.7% formaldehyde for 15 min, washed three times with PBS and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Mouse monoclonal anti-snail, anti-vimentin, and Alex Fluor®568 goat anti-mouse IgG (Millipore Inc.) were used as primary and secondary antibodies, respectively. Images were obtained using a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Jena, Germany).

Xu Z., Zhou Z., Zhang J., Xuan F., Fan M., Zhou D., Liuyang Z., Ma X., Hong Y., Wang Y., Sharma S., Dong Q, & Wang G. (2020). Targeting BMI-1-mediated epithelial–mesenchymal transition to inhibit colorectal cancer liver metastasis. Acta Pharmaceutica Sinica. B, 11(5), 1274-1285.

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Western Blotting Analysis of Protein Expression

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Western blotting was performed as previously described [48 (link)], using anti-Gankyrin (1:1000), anti-vimentin(1:1000), anti-PTEN(1:1000), anti-P21(1:1000), anti-cyclin D1(1:1000), anti-c-myc(1:1000), anti-MMP7 (1:1000) antibodies (Santa Cruz Biotech., Santa Cruz, CA), anti-E-cadherin(1:5000), anti-fibronectin (1:5000)antibodies (BD Biosciences, San Jose, CA), anti-p-Akt^ser473(1:1000), anti-Akt (1:2000), anti-p-GSK-3b^ser9 (1:1000), anti-GSK-3b (1:1000), anti-β-catenin (1:1000) antibodies (Cell Signaling, Danvers, MA). The membranes were stripped and re-probed with anti-α-tubulin (1:3000) antibody or anti-β-actin (1:5000) (Cell Signaling) as a loading control. The antibody concentration was according to the recommendation of respective specification.

He F., Chen H., Yang P., Wu Q., Zhang T., Wang C., Wei J., Chen Z., Hu H., Li W, & Cao J. (2016). Gankyrin sustains PI3K/GSK-3β/β-catenin signal activation and promotes colorectal cancer aggressiveness and progression. Oncotarget, 7(49), 81156-81171.

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Proximity Ligation Assay for Protein-Protein Interactions

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Proximity ligation assay (PLA) was performed using Duolink^TM In Situ Probe Anti-Mouse PLUS, Anti-Rabbit MINUS, Wash Buffers (Fluorescence), Reagents Green, and Mounting medium with DAPI, according to manufacturer’s instructions (Sigma-Aldrich). Primary antibodies combinations used were anti-RUNX3 rabbit (1:400) (Cell Signaling Technology, D6E2) and anti-MYC mouse (1:400) (OriGene, OTI3F2) monoclonal antibodies; anti-RUNX3 (1:400) (Cell Signaling Technology, D9K6L) and anti-MYC (1:400) (Cell Signaling Technology, D84C12); anti-RUNX3 (1:400) (Cell Signaling Technology, D9K6L) and anti-GSK3B (1:400) (Cell Signaling Technology, D5C5Z); anti-RUNX3 (1:400) (Cell Signaling Technology, D9K6L) and FBXW7 (1:400) (Proteintech, 55290-1-AP); anti-RUNX3 (1:400) (Cell Signaling Technology, D9K6L), and MAX (1:400) (abcam, ab199489); RUNX3 (1:400) (Cell Signaling Technology, D9K6L) and MIZ-1 (1:400) (Cell Signaling Technology, D7E8B). The signals were visualized with Zeiss LSM880 confocal microscope and analyzed with Zeiss Zen (Blue) imaging software.

Oei V., Chuang L.S., Matsuo J., Srivastava S., Teh M, & Ito Y. (2023). RUNX3 inactivates oncogenic MYC through disruption of MYC/MAX complex and subsequent recruitment of GSK3β-FBXW7 cascade. Communications Biology, 6, 689.

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Western Blot Analysis of Signaling Pathways

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Following the different treatments, as indicated in the figure legends, the cells were lysed, and the protein concentration of each lysate was determined. Subsequently, aliquots (15 μg) of the lysates were subjected to western blot analysis. The following antibodies were used: anti-phospho-AKT (Ser473), anti-S6, anti-phospho-S6 (Ser235/236), anti-GSK3B, anti-phospho-GSK3B (Ser9), anti-phospho-70S6K, anti-phospho-p70S6K (Thr421/Ser424), anti-4EBP1, anti-phospho-4EBP1 (Ser65), anti-PI3K-p110α, and anti-PI3K-p110β from Cell Signaling Technology (Danvers, MA, United States), and anti-AKT from Santa Cruz Biotechnology (Dallas, TX, United States). All of the dilution and preparation of antibodies were performed according to manufacturer’s datasheet.

Chen I.C., Hsiao L.P., Huang I.W., Yu H.C., Yeh L.C., Lin C.H., Wei-Wu Chen T., Cheng A.L, & Lu Y.S. (2017). Phosphatidylinositol-3 Kinase Inhibitors, Buparlisib and Alpelisib, Sensitize Estrogen Receptor-positive Breast Cancer Cells to Tamoxifen. Scientific Reports, 7, 9842.

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Immunoblotting Assays: Antibody Characterization

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The following antibodies were used for immunoblotting assays: anti-AKT (#9272), anti-ERK1/2 (#4695), anti-GSK-3B (#9315), anti-MCL-1 (#4572), anti-SYK (#2172) and anti-LYN (#4576) were purchased from Cell Signaling (Danvers, MA, USA). Anti-XIAP (#610762) was purchased from BD Bioscience (San Jose, CA, USA). Anti-HSP90B (#837159A) and anti-GAPDH (#TAB1001) were purchased from Thermo Fisher Scientific (San Jose, CA, USA). Anti-BCL-2 (#k2206) was purchased from Santa Cruz Biotechno-logy (Santa Cruz, CA, USA). Annexin V-fluorescein isothiocyanate and 7-Aminoactinomycin D were obtained from BD Bioscience. The propidium iodide and InnoCyte Flow Cytometric Cytochrome c Release kit were purchased from EMD Millipore (Kankakee, IL, USA). PU-H71 and PU-H71-conjugated agarose beads were obtained from Dr Gabriela Chiosis and were prepared as previously reported.^{17 (link)} Amaxa Solution V Nucleofector kit was purchased from Amaxa (Cologne, Germany). Pools of siRNA against human BTK, AKT and HSP90 were customarily designed and purchased from Thermo Scientific (Waltham, MA, USA).

Guo A., Lu P., Lee J., Zhen C., Chiosis G, & Wang Y. (2017). HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment. Oncogene, 36(24), 3441-3449.

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