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Phospho gsk3α

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Phospho-GSK3α is a primary antibody that detects endogenous levels of GSK3α only when phosphorylated at Ser21. GSK3α is a serine/threonine protein kinase involved in the regulation of various cellular processes.

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Lab products found in correlation

Phospho gsk3β, by Cell Signaling Technology (3 mentions) Total gsk3β, by Cell Signaling Technology (2 mentions) Total gsk3α, by Cell Signaling Technology (2 mentions) Anti β actin, by Merck Group (1 mentions) Phospho β catenin, by Cell Signaling Technology (1 mentions) Anti β catenin, by Abcam (1 mentions) Pka c α, by Cell Signaling Technology (1 mentions) Phosphor creb, by Cell Signaling Technology (1 mentions) Anti non phospho β catenin, by Cell Signaling Technology (1 mentions) Total creb, by Cell Signaling Technology (1 mentions) Parp1, by Cell Signaling Technology (1 mentions) Anti cd31, by Abcam (1 mentions) Alexa conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Mg132, by Merck Group (1 mentions) Eif4e, by Cell Signaling Technology (1 mentions) Phospho cyclin d1, by Cell Signaling Technology (1 mentions) Pnu 74654, by Santa Cruz Biotechnology (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions) Alexa fluor 680, by Thermo Fisher Scientific (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)

Spelling variants of this product

GSK3α (24 citations) Phospho-GSK3α/β (16 citations) Phospho-GSK3α/β (Ser21/9) (14 citations) Anti-phospho-GSK3α/β (Ser21/9) (10 citations) Phospho-Ser21-GSK3α (5 citations) Rabbit anti-Phospho-GSK3α (Ser21) antibody (3 citations) Phospho-GSK3α/β (Ser21/Ser9) (2 citations) Anti-phospho (Ser 21/9) GSK3α/β (2 citations) Phospho-GSK3α/βS21/9 (2 citations)

6 protocols using phospho gsk3α

1

Signaling Pathway Analysis Protocol

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Western blot, cell fractionation and Co-IP analysis were carried out as described in our previous publications.23 (link),26 (link),29 (link) The following primary antibodies were used: anti-ITGA9, anti-CD31 (Abcam, Cambridge, MA); anti-β-catenin, anti-non-phospho-β-catenin, phospho-β-catenin (Ser33/37/Thr41, Thr41/Ser45, Ser675), phospho-GSK3α (Ser21), phospho-GSK3β (Ser9), total GSK3α, total GSK3β, phosphor-CREB (Ser133), total CREB, integrin-linked kinase (ILK), PKA-Cα, and PARP-1 (Cell Signaling Technology, Beverly, MA); anti-PKA RIIα regulatory unit (Santa Cruz Biotechnology, Dallas, TX); anti-β-actin (Sigma, St. Louis, MO).
Wang Z., Li Y., Xiao Y., Lin H.P., Yang P., Humphries B., Gao T, & Yang C. (2019). Integrin α9 depletion promotes β-catenin degradation to suppress triple negative breast cancer tumor growth and metastasis. International journal of cancer, 145(10), 2767-2780.
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2

Wnt/β-catenin Inhibitors and Their Effects

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Wnt/β-catenin inhibitors, iCRT3 and PNU-74654, were from Santa Cruz Biotechnology (Santa Cruz, CA). MG-132, was obtained from Sigma-Aldrich Chemical Co., Inc. (St. Louis, MO). Vectashield mounting medium was from Vector Laboratories Inc. (Burlingame, CA). siRNA for β-catenin was from Dharmacon (Lafayette, CO). Rabbit antibodies to cyclin D1, GSK3α, GSK3β, phospho GSK3α, phospho GSK3β, phospho cyclin D1, eukaryotic initiation factor 4E (eIF4E) and Alexa-conjugated secondary antibodies were from Cell Signaling Technology Inc. (Beverly, MA). Anti-eIF4E (phospho S209) antibody was from Abcam Inc. (Cambridge, MA). Extracellular domain of receptor for advanced glycation end products (sRAGE) was prepared as described (11 (link)). All other reagents were provided as described (11 (link),12 ). Scrambled siRNA was used as negative control for all siRNA experiments as described (11 (link),12 ). PolyP-70 was a generous gift from Dr. James Morrissey (University of Illinois, Urbana). Platelets releasates were prepared by activation of human platelets with TRAP as described (21 (link)).
Hassanian S.M., Ardeshirylajimi A., Dinarvand P, & Rezaie A.R. (2016). Inorganic polyphosphate promotes cyclin D1 synthesis through activation of mTOR/Wnt/β-catenin signaling in endothelial cells. Journal of thrombosis and haemostasis : JTH, 14(11), 2261-2273.
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3

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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4

Protein Expression Analysis of Cell Lysates

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After desired treatments, cells were lysed with RIPA buffer spiked with a fresh protease and phosphatase cocktail (Thermo Scientific, #78442) and sonicated. Protein concentrations were quantified using the Pierce BCA assay kit (Thermo Fisher, #23225). 80–120μg of protein for each sample was loaded onto SDS-PAGE gels, and then transferred onto PVDF membranes. The blots were incubated with the following antibodies: desmocollin 1 (sc-398590, RRID: AB_2894905), desmoglein 2 (sc-80663, RRID: AB_2093438), plakophilin (sc-33636, RRID: AB_2164139), connexin 26 (sc-7261, RRID: AB_2110895) and cFOS (sc-52, RRID: AB_2106783) from Santa Cruz; ER-α (#8644, RRID: AB_2617128), HA (#3724, RRID: AB_1549585), Non-phospho-β-catenin (#19807, RRID: AB_2650576), Histone H3 (#4499, RRID: AB_10544537), AIF (#5318, RRID: AB_10634755), GSK3β (Ser9, #5558, RRID: AB_10013750), phospho-GSK3α (Ser21, #9316, RRID: AB_659836), GSK3β (#12456, RRID: AB_2636978) and GSK3α (#4337, RRID: AB_10859910) from Cell Signaling Technology; β-catenin (#610154, RRID: AB_397555) from BD; Tubulin (T6557, RRID: AB_477584) and connexin 43 (C6219, RRID: AB_476857) from Sigma Aldrich; and TIMP3 (ab39184, RRID: AB_2204971) from Abcam.
Li Z., Wu Y., Yates M.E., Tasdemir N., Bahreini A., Chen J., Levine K.M., Priedigkeit N.M., Nasrazadani A., Ali S., Buluwela L., Arnesen S., Gertz J., Richer J.K., Troness B., El-Ashry D., Zhang Q., Gerratana L., Zhang Y., Cristofanilli M., Montanez M.A., Sundd P., Wallace C.T., Watkins S.C., Fumagalli C., Guerini-Rocco E., Zhu L., Tseng G.C., Wagle N., Carroll J.S., Jank P., Denkert C., Karsten M.M., Blohmer J.U., Park B.H., Lucas P.C., Atkinson J.M., Lee A.V, & Oesterreich S. (2022). Hotspot ESR1 mutations are multimodal and contextual modulators of breast cancer metastasis. Cancer research, 82(7), 1321-1339.
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5

Western Blot Analysis of Signaling Pathways

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Neutrophils were lysed in 1X NP-40 lysis buffer supplemented with protease inhibitor cocktail. Lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Primary antibodies used were as follows: Phospho-AKT (Ser473) (Catalog no. 4-60T), total AKT (Catalog no. 9272S), Phospho-GSK3α (Ser21) (Catalog no. 9316T), total GSK3α (Catalog no. 9338S), Phospho-GSK3β (Ser9) (Catalog no. 5558S), total GSK3β (Catalog no. 9315S), Phospho-p70 S6 Kinase (Thr389) (Catalog no. 9234S), total p70S6Kinase (Catalog no. 2708), Phospho-4E-BP1 (Thr37/46) (Catalog no. 2855), total 4EBP-1 (Catalog no. 9452) (Cell Signaling Technology), Anti-PKCα (S657) (Catalog no. ab180848), total PKCα (Catalog no. ab32376), Glut1 (Catalog no. ab115730), Glut3 (Catalog no. ab191071, and anti-mouse β-actin (Abcam, Cambridge, MA). Protein bands were detected using an enhanced chemiluminescence detection system (ThermoScientific) and developed with a FluorChem E imager (ProteinSimple, San Jose CA). For densitometry analysis, protein band corresponding to proteins of interest were analyzed by ImageJ software.
Jawale C.V., Ramani K., Li D.D., Coleman B.M., Oberoi R.S., Kupul S., Lin L., Prokopienko A.J., Desai J.V., Delgoffe G.M., Lionakis M.S., Bender F.H., Nolin T.D., Gaffen S.L, & Biswas P.S. (2020). Restoring glucose uptake rescues neutrophil dysfunction and protects against fatal bloodstream fungal infections in kidney disease. Science translational medicine, 12(548), eaay5691.
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6

Investigating Wnt Pathway Modulation in CuB-Treated Cancer Cells

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Whole cell lysates of A549 and H1299 cells at 24 h of CuB treatment were prepared using the RIPA-lysis buffer (Millipore, Billerica, MA). Protein extraction from tumor samples was also performed using RIPA-lysis buffer. Proteins were resolved on 10–12% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore). After incubation in blocking buffer (5% skimmed milk or 2% BSA in TBST) for 1 h, the membranes were incubated with primary antibodies specific for Wnt3/3a (Abcam: ab172612), Fzd-7 (Sigma-aldrich: AV41251), phospho-GSK-3α (Cell Signaling Technology: 9337), GSK-3α (Abcam: ab40870), phospho-GSK-3β (Santa Cruz: SC-11757), GSK-3β (Abcam: ab18893), β-catenin (Invitrogen: 71-2700), TCF-1 (Life Technologies: A13969), MMP-2 (Cell Signaling Technology: 4022S), E-cadherin (Santa cruz: SC-8426), COX-2 (Millipore: AB5118), MYC (Millipore: 06-340), Cyclin D1 (Santa cruz: SC-8396), Survivin (Cell Signaling Technology: 2808S), VEGF (Santa cruz: S C-53462), Vimentin (Santa cruz: SC-6260), PCNA (Santa cruz: SC-7907) and β-actin (Cell Signaling Technology: 4970L). The blots were then incubated with specific HRP-conjugated secondary antibodies and bands were visualized using ECL (Millipore) on ImageQuant LAS4010 chemiluminescence detection system (GE Healthcare, Amarsham, UK). The band intensities were quantified using ImageJ 1.46r software.
Shukla S., Sinha S., Khan S., Kumar S., Singh K., Mitra K., Maurya R, & Meeran S.M. (2016). Cucurbitacin B inhibits the stemness and metastatic abilities of NSCLC via downregulation of canonical Wnt/β-catenin signaling axis. Scientific Reports, 6, 21860.
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