Gsk3β

Manufactured by ABclonal

Sourced in United States, China

GSK3β is a serine/threonine protein kinase that plays a key role in the regulation of various cellular processes, including glycogen metabolism, cell division, and cell survival. It is an important component of the Wnt/β-catenin signaling pathway and is involved in the phosphorylation of several substrates, including glycogen synthase, tau, and β-catenin.

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Lab products found in correlation

P gsk 3β, by ABclonal (4 mentions) P ampk α1, by ABclonal (2 mentions) Ampk α1, by ABclonal (2 mentions) Cox 2, by ABclonal (2 mentions) P akt, by Cell Signaling Technology (2 mentions) Nqo 1, by ABclonal (2 mentions) β actin, by ABclonal (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Vimentin, by Santa Cruz Biotechnology (1 mentions) N cadherin, by ABclonal (1 mentions) β catenin, by ABclonal (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) E cadherin, by ABclonal (1 mentions) N cadherin, by Proteintech (1 mentions) Dach1, by ABclonal (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions) Ripa buffer, by Solarbio (1 mentions) E cadherin, by Immunoway (1 mentions) Phosphatase inhibitor, by Solarbio (1 mentions)

8 protocols using gsk3β

Antibody Optimization for Western Blot

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Antibodies for western blot analyses and the final working dilutions were as follows: rabbit polyclonal antibodies to α-SMA (dilution 1 : 1000; ABclonal, MA, USA), β-catenin (dilution 1 : 1000; ABclonal, MA, USA), vimentin (dilution 1 : 500; Santa Cruz, TX, USA), N-cadherin (dilution 1 : 1000; ABclonal, MA, USA), E-cadherin (dilution 1 : 1000; ABclonal, MA, USA), Wnt-3 (dilution 1 : 1000; ABclonal, MA, USA), p-GSK3β (dilution 1 : 1000; ABclonal, MA, USA), GSK3β (dilution 1 : 1000; ABclonal, MA, USA), β-actin (dilution 1 : 1000, Santa Cruz, TX, USA).
p65 (dilution 1 : 1000; GeneTex, CA, USA), and fibrillin (dilution 1 : 1000, ABclonal, MA, USA). All HRP-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Inc. (PA, USA).

Chung P.C., Hsieh P.C., Lan C.C., Hsu P.C., Sung M.Y., Lin Y.H., Tzeng I.S., Chiu V., Cheng C.F, & Kuo C.Y. (2020). Role of Chrysophanol in Epithelial-Mesenchymal Transition in Oral Cancer Cell Lines via a Wnt-3-Dependent Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2020, 8373715.

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Western Blot Analysis of Cell Signaling

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Cell lysates were prepared in RIPA buffer (#R0020; Solarbio) with protease inhibitors (1 mM PMSF, #P0100; Solarbio), separated with SDS‐PAGE and transferred onto PVDF membranes, which were blocked in 5% milk, and then incubated with the primary antibody overnight at 4°C followed with secondary antibody (mouse IgG, #5220‐0341; Seracare; rabbit IgG, #5220‐0336; Seracare) for 2 h. The primary antibodies were used as follows: β‐Catenin (#8480s; CST), E‐cadherin (#YM0207; IMMUNOWAY), N‐cadherin (#22018; PROTEINTECH), DACH1 (#A3823; ABclonal), CEBPA (#8178S; CST), p65 (#8242; CST), p‐p65 (#3033; CST), AKT (#4691; CST), p‐AKT (#4060; CST), LATS2 (#5888; CST), ERK1/2 (#4695; CST), p‐ERK1/2 (#4370; CST), p38 (#8690; CST), p‐p38 (#4511; CST), BRAF (#14814; CST), c‐Jun (#9165; CST), JunB (#3753; CST), c‐FOS (#2250; CST), TPO (#ab203057; Abcam), NIS (#ab242007; Abcam), PAX8 (#ab97477; Abcam), GSK3β (#A2081; ABclonal), p‐GSK3β (#AP0039; ABclonal).

Zhang P., Guan L., Sun W., Zhang Y., Du Y., Yuan S., Cao X., Yu Z., Jia Q., Zheng X., Meng Z., Li X, & Zhao L. (2024). Targeting miR‐31 represses tumourigenesis and dedifferentiation of BRAFV600E‐associated thyroid carcinoma. Clinical and Translational Medicine, 14(5), e1694.

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Protein Expression Analysis in Macrophages

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Total protein was extracted from RAW264.7 macrophages and primary peritoneal macrophages using radio immunoprecipitation lysis buffer (RIPA, Solarbio, Beijing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors (Solarbio, Beijing, China). The protein was collected, and its concentration was measured using a Pierce BCA protein assay kit (Thermo, Rockford, IL, USA). Equal amounts of proteins were separated by electrophoresis on SDS-PAGE gels and subsequently transferred onto PVDF membranes. Blocking was with TBST containing non-fat milk powder, the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies against NRF2, p-AKT, AKT, iNOS (Cell Signaling Technology, Danvers, MA, USA), and HO-1, NQO-1, COX-2, p-GSK-3β, GSK-3β, p-AMPK-α1, AMPK-α1, p-ERK, EKR, p-JNK, JNK, p-P38, P38, p-NF-κB p65, NF-κB p65, and GAPDH (ABclonal, Wuhan, China) were diluted to a ratio of 1:2000. Before adding secondary antibodies (goat anti-rabbit or goat anti-mouse) diluted to a concentration of 1:10,000 (ABclonal, Wuhan, China), the membranes were washed four times with TBST for 15 min each time. Membranes were visualized using an Amersham Imager 600 (a gel imaging system from GE Co., Fairfield, CT, USA) after applying enhanced chemiluminescence. For detailed information of WB procedures, refer to our previous study [50 (link)].

Chen G., Wen D., Shen L., Feng Y., Xiong Q., Li P, & Zhao Z. (2023). Cepharanthine Exerts Antioxidant and Anti-Inflammatory Effects in Lipopolysaccharide (LPS)-Induced Macrophages and DSS-Induced Colitis Mice. Molecules, 28(16), 6070.

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Flnc Silencing Modulates Wnt/β-Catenin Signaling

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To verify the activity of Wnt/β-catenin signaling, Flnc-silenced and control cells were treated with 10 mM LiCl (Sigma) and 200 ng wnt3a (Sigma) for 24 h in differentiation medium. To verify activity of wnt signaling, Flnc-silenced cells were treated with 1-AKP (Selleck, Houston, TX, USA) or DMSO for 48 h. The following primary antibodies were used: anti-MyHC (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-MyoD (Santa Cruze, CA, USA), anti-MyoG (Biorbyt, Cambridge, UK); Flnc (ZenBio, Chengdu, China); GSK3β (ABclonal, Wuhan, China); Axin1 (Cell Signaling Technology, Boston, MA, USA); β-catenin (Cell Signaling Technology); Anti-Dvl2 (Abcam, Cambridge, UK); anti-Ubiquitin (Abcam); anti-Axin1 (Bioss, Beijing, China); anti-H3 (ABclonal, Wuhan, China); and GAPDH (ZenBio). The following secondary antibodies were used: mouse anti-rabbit horseradish peroxidase (HRP) and goat anti-mouse HRP from ZenBio Biology (ZenBio).

Han S., Cui C., Zhao X., Zhang Y., Zhang Y., Zhao J., Shen X., He H., Wang J., Ma M., Li D., Zhu Q, & Yin H. (2021). Filamin C regulates skeletal muscle atrophy by stabilizing dishevelled-2 to inhibit autophagy and mitophagy. Molecular Therapy. Nucleic Acids, 27, 147-164.

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Protein Expression Analysis in Metformin and EGCG Treated Cells

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Cells were treated the same as 4.5. and then washed twice with ice-cold PBS and were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) after the treatment with metformin or/and EGCG co-cultured with MG. The Supernatant was collected, and concentration was determined by BCA protein assay kit (Solarbio, Beijing, China). The same amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk for 2 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies Bcl-2 (12789-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Caspase-3 (A11040, ABclonal), Cytochrome C (A4912, ABclonal), GSK-3β (A2081, ABclonal), BACE-1 (AF6273, Beyotime), APP (A17911, ABclonal), and β-actin (AC038, ABclonal). The membranes were washed with 1% TBST three times, and then incubated with secondary antibodies (1:3000 dilution) for 1 h at room temperature. Finally, the bands were visualized using an ECL kit (Tanon, Shanghai, China).

Zhang D., He X., Wang T., Xing Y., Xiu Z., Bao Y, & Dong Y. (2024). Synergistic Effect of Flavonoids and Metformin on Protection of the Methylglyoxal-Induced Damage in PC-12 Neuroblastoma Cells: Structure–Activity Relationship and Potential Target. Molecules, 29(10), 2306.

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Immunoblotting of Intracellular Signaling

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The detailed experimental procedures can be found in our previous study [35 (link)]. Antibodies used included p-AKT, AKT, NRF2, and iNOS from Cell Signaling Technology (Danvers, MA, USA) and NQO-1, HO-1, COX-2, p-AMPK-α1, AMPK-α1, p-GSK-3β, GSK-3β, β-actin, and goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody from ABclonal (Wuhan, China).

Wen D., Meng C., Feng Y., Shen L., Liu Y., Sun W., Chen G, & Wu C. (2024). Syringaldehyde Exhibits Antibacterial and Antioxidant Activities against Mycobacterium marinum Infection. Microorganisms, 12(2), 348.

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Investigating Molecular Mechanisms in Neurodegeneration

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TPPU was purchased from R&D Systems, Inc (Minneapolis, USA). The primary antibodies COX-2, Grp78, IKKβ, p-IKKβ, p65, p-p65, caspase 9, cleaved-caspase 9, caspase 3, cleaved-caspase 3, and p-GSK3β (Ser9) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). The primary antibodies Tau, p-Tau (Thr205), p-Tau (Ser262), p-Tau (Ser404), HO-1, GSK3β, and sEH were purchased from ABclonal (Wuhan, China). The primary antibodies GAPDH, Aβ, Bax, IL-6, and GFAP were purchased from Proteintech (Wuhan, China). The primary antibodies Bcl-2, Iba-1, Nrf2, and Keap1 were purchased from Abcam (Cambridge, United Kingdom). Aβ_1–42 (Aβ) was purchased from Sigma Aldrich (St. Louis, Missouri, USA).

Sun C.P., Zhang X.Y., Zhou J.J., Huo X.K., Yu Z.L., Morisseau C., Hammock B.D, & Ma X.C. (2021). Inhibition of sEH via stabilizing the level of EETs alleviated Alzheimer’s disease through GSK3β signaling pathway. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 156, 112516.

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Sildenafil Citrate Cytotoxicity Assay

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Sildenafil citrate (purity > 98%) was purchased from Topscience. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Lipopolysaccharides (LPS) (Cat# L2880) was purchased from Sigma-Aldrich. Antibodies against CDK5 (Cat# A5730), GSK3β (Cat# A2081), and phospho-GSK3B-Y216 (Cat# AP0261) were obtained from ABclonal Technology. CDK5-Phospho-Tyr15 (Cat# YP0380) was purchased from Immunoway (Plano, Texas, USA). All other reagents were obtained from Sigma-Aldrich unless otherwise specified. The validation of all primary antibodies for the species are available from the manufacturer’s website.

Fang J., Zhang P., Zhou Y., Chiang C.W., Tan J., Hou Y., Stauffer S., Li L., Pieper A.A., Cummings J, & Cheng F. (2021). Endophenotype-based in silico network medicine discovery combined with insurance record data mining identifies sildenafil as a candidate drug for Alzheimer’s disease. Nature aging, 1(12), 1175-1188.

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