Anti gsk3α

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-GSK3α is a laboratory reagent that binds to and detects the presence of the GSK3α protein. GSK3α is a serine/threonine protein kinase involved in the regulation of various cellular processes. This product can be used to study the expression and activity of GSK3α in experimental settings.

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7 protocols using anti gsk3α

Western Blot Analysis of AKT/FoxO1/GSK3 Signaling

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Whole tissue protein extracts were prepared with NP-40 buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40) containing protease inhibitors (SigmaFast, Sigma-Aldrich). Western blot analysis was carried out by standard methods[11 (link)]. Anti-phospho-AKT (Ser473) (#4058), anti-AKT1 (#2938), anti-phospho-FoxO1 (Ser256) (#9461), anti-FoxO1 (#2880), anti-phospho-GSK3-α/β (Ser21/9) (#9331), anti-GSK3-α (#9338), anti-GSK3-β (#9332) were purchased from Cell Signaling Technology (Danver, MA).

Malanga D., Belmonte S., Colelli F., Scarfò M., De Marco C., Oliveira D.M., Mirante T., Camastra C., Gagliardi M., Rizzuto A., Mignogna C., Paciello O., Papparella S., Fagman H, & Viglietto G. (2016). AKT1E17K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer. PLoS ONE, 11(2), e0147334.

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Western Blot Analysis of Signaling Proteins

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Western blot analysis was performed as described previously [34 (link)]. The following antibodies were used in this study: anti-AKT (Cell Signaling, 2920S), anti-phospho-ERK(CellSignaling,4370S), anti-phospho-AKT(Ser473) (Epitomics,2118-1), anti-GAPDH(Santa Cruz Biotechnology, SC-32233), PARP(Santa Cruz Biotechnology, SC-8007), anti-caspase-3(Cell Signaling, 9665S), anti-actin(Sigma, A4700), anti-cytochrome C(Santa Cruz Biotechnology, SC-13156), anti-Tom20(Santa Cruz Biotechnology, SC-136211), anti-Mcl-1(Santa Cruz Biotechnology, SC-819), anti-GFP(Sigma, G1544), anti-ERK(Cell Signaling, 4695S), anti-TNFAIP1(proteintech, 60327), anti-LIN28(proteintech,11724), anti-GBP1(proteintech, 15303), anti-Bcl-2(proteintech, 12789), anti-Bxl-xl(proteintech, 26967), anti-GSK3α(Cell Signaling, 9338), anti-phospho-GSK3α(ser21)(Boster, P03152), anti-GSK3β(Cell Signaling, 12456), anti-phospho-GSK3β(ser9)( Cell Signaling, 5558).

Shuang W., Hou L., Zhu Y., Li Q, & Hu W. (2017). Mcl-1 stabilization confers resistance to taxol in human gastric cancer. Oncotarget, 8(47), 82981-82990.

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Western Blot Analysis of Kinase Signaling

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Equal amounts of protein were loaded onto gels subjected to SDS-PAGE (sodium dodecyl sulfate-poly-acrylamide gel electrophoresis), followed by transfer to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk for one hour at room temperature and then incubated with the primary antibody: anti-PKA (1:2000, rabbit, Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated PKA (Thr-197) (1:2000, rabbit, Abcam), anti-GSK-3α (1:1000, rabbit, Cell Signaling Technology), anti-GSK-3β (1:1000, rabbit, Cell Signaling Technology), or anti-phosphorylated GSK-3α/β (Ser-21/Ser-9) (1:1000, rabbit, Cell Signaling Technology), overnight at 4 °C. The membrane was then incubated with the appropriate horseradish peroxidase conjugated secondary antibody diluted in 1% bovine serum albumin in Tris-Buffered Saline (TBS) supplemented with Tween-20 for two hours at room temperature. The proteins were visualized by ECL clarity reagents (Bio-Rad) or enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ, USA). The blot images were collected using the Bio-Rad ChemiDoc MP imaging system (Bio-Rad), and the intensity of the bands quantified by Image Lab software (Bio-Rad).

Jiang A., Wang L., Lu J.Y., Freeman A., Campbell C., Su P., Wong A.H, & Liu F. (2021). Sex Differences in Dopamine Receptor Signaling in Fmr1 Knockout Mice: A Pilot Study. Brain Sciences, 11(11), 1398.

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Protein Expression and Signaling Analysis

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Whole cell lysates were prepared in M-PER buffer (Thermo, Waltham, MA), resolved by SDS-PAGE and blotted with indicated antibodies. The following antibodies were used in this study: Anti-HA, anti-FLAG, anti-phospho-β-catenin (Ser33/37/Thr41), anti-β-catenin, anti-GSK3α, anti-β-TrCP, anti-α-Tubulin, anti-GSK3β, anti-FAK, anti-phospho-FAK (Tyr397), anti-PYK2 and anti-phospho-PYK2 (Tyr402) (Cell Signaling); anti-FLAG M2 (Sigma); anti-ubiquitin and anti-β-catenin (BD Bioscience, San Jose, CA); anti-GSK3, anti-c-Myc, anti-PYK2, anti-β-TrCP, anti-phospho-GSK3β (Tyr216) and anti-β-Actin (Santa Cruz Biotechnology, Dallas, TX); anti-phospho-GSK3 (Tyr279/Tyr216) (Millipore). anti-GSK3β for IP was purchased from Abcam (Cambridge, MA) and Bethyl Laboratories (Montgomery, TX). SuperSignal West Pico Chemiluminescent Substrate and SuperSignal Western Blot Enhancer (Thermo) were used to enhance western signal when needed. PF-562271 was purchase from MedKoo Biosciences (Chapel Hill, NC) and Selleck Chemicals (Houston, TX). Gelucire is a gift from Gelucire-4414.html">Gattefosse (Paramus, NJ). MG-132 was obtained from Sigma.

Gao C., Chen G., Kuan S.F., Zhang D.H., Schlaepfer D.D, & Hu J. (2015). FAK/PYK2 promotes the Wnt/β-catenin pathway and intestinal tumorigenesis by phosphorylating GSK3β. eLife, 4, e10072.

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Flow Cytometry and Western Blot Analysis of Wnt Pathway

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The antibodies used for blast cell identification by Flow Cytometry (FACSCanto II, Becton Dickinson, Rutherford, NJ, USA) were: anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC all from Miltenyi Biotech (Bergsch gradbach, Germany). For Western blot analysis, anti-β-catenin, anti- Ser675-phospho-β-catenin, anti-Ser33/37/Thr41-phospho-β-catenin, anti-non-phopho-β-catenin, anti-GSK-3β, anti-Ser9-phospho-GSK-3β, anti-GSK-3α, anti-Ser21-phospho-GSK-3α anti-Histone H3 antibodies and Alexa 488-conjugated secondary antibodies were from Cell Signaling (Danvers, MA, USA); anti-GAPDH, and HRP-conjugated secondary antibodies against mouse or rabbit were from Sigma-Aldrich (Darmstadt, Germany). Wnt modulators used for proliferation and vitality assays, i.e., Wnt-3a, PNU-74654, Niclosamide, IWP-2, Lithium Chloride (LiCl), and AR-A014418, were all purchased from Sigma-Aldrich. For the analysis of cell death, Propidium iodide (PI) and FITC-conjugated Annexin V were from Miltenyi Biotechnology. CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS, Eden Prairie, MN, USA) was from Promega (Promega, Milano, Italy). Cytarabine (Ara-C) and Idarubicin (Ida) were provided by the Pharmacy Unit of the University Hospital of Verona.

Takam Kamga P., Dal Collo G., Cassaro A., Bazzoni R., Delfino P., Adamo A., Bonato A., Carbone C., Tanasi I., Bonifacio M, & Krampera M. (2020). Small Molecule Inhibitors of Microenvironmental Wnt/β-Catenin Signaling Enhance the Chemosensitivity of Acute Myeloid Leukemia. Cancers, 12(9), 2696.

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Western Blot Analysis of LV Tissue

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LV tissue was homogenized in 1X lysis buffer (Cell Signaling# 9803) with a protease and phosphatase inhibitor cocktail. After homogenization, samples were centrifuged at 15,000 g for 15 minutes at 4°C and supernatant transferred to fresh tubes. Protein concentration in the supernatant was quantified with the bicinchoninic acid protein assay (Pierce# 23225). Equal amounts of proteins were subjected to SDS-PAGE and subsequently were transferred to nitrocellulose membranes. A primary antibody incubations was performed at 1:1,000 dilution for anti-GSK-3α (Cell Signaling# 5676), phospho-GSK-3α (Cell Signaling #9316), Bax (Cell Signaling #2772), Bcl-xL (Cell Signaling #2764), VDAC (Cell Signaling #4661), 1:100 for Cyclin E (Santa Cruz #sc-481) and phospho-Cyclin E1 (Santa Cruz sc-12917-R) and 1:10,000 for GAPDH (Fitzgerald #10R-G109a). All incubations were done at 4°C, overnight. The secondary antibody used was Alexa Fluor 680 (Molecular Probes), at 1:3,000 dilution for 1 hour at room temperature. Membranes were scanned with the Odyssey Infrared Imaging System (LI-COR).

Ahmad F., Singh A.P., Tomar D., Rahmani M., Zhang Q., Woodgett J.R., Tilley D.G., Lal H, & Force T. (2019). Cardiomyocyte-GSK-3α promotes mPTP opening and heart failure in mice with chronic pressure overload. Journal of molecular and cellular cardiology, 130, 65-75.

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Immunohistochemical Analysis of GSK3 in DRGs

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Paraffin sections of DRGs (L4 to L6) from eight-week-old cre⁺GSK3βlox/lox and cre⁺GSK3βwt/wt mice were blocked and incubated overnight at 4°C with the following antibodies: anti-GSK3β (1:100; Cell Signaling), anti-GSK3α (1:100; Cell Signaling), and anti-GFP (1:1000; Clontech, Lisboa, Portugal). Subsequently, incubation was performed with the respective fluorescent secondary antibodies and slides were mounted in vectashield with 4′,6-diamidino-2-phenylindole (DAPI.

Liz M.A., Mar F.M., Santos T.E., Pimentel H.I., Marques A.M., Morgado M.M., Vieira S., Sousa V.F., Pemble H., Wittmann T., Sutherland C., Woodgett J.R, & Sousa M.M. (2014). Neuronal deletion of GSK3β increases microtubule speed in the growth cone and enhances axon regeneration via CRMP-2 and independently of MAP1B and CLASP2. BMC Biology, 12, 47.

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