Mouse anti gsk3β

Manufactured by Cell Signaling Technology

Sourced in United States

Mouse anti-GSK3β is a primary antibody that recognizes the glycogen synthase kinase 3 beta (GSK3β) protein, a serine/threonine protein kinase involved in the regulation of various cellular processes. This antibody is suitable for use in western blotting and other immunodetection applications to identify and study the GSK3β protein.

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GSK3β (613 citations) Anti-GSK3β (194 citations) Mouse anti‐GSK3β (clone 3D10, (2 citations)

6 protocols using mouse anti gsk3β

Western Blot Analysis of Cellular Signaling

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Western blot analysis was carried out as described previously (15 (link)). Cells or kidney tissues were lysed in cell lysis buffer (Cell Signaling Technology) and homogenized, followed by centrifugation for 10 minutes at 15,000g. Protein concentrations were determined using the bicinchoninic acid protein assay. Electrophoresis was conducted with 10% or 12.5% running gels and 5% stacking gels. Proteins were transferred onto a nitrocellulose membrane. After blockade with 5% skim milk in Tris buffered saline–0.1% Tween 20, membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti–GSK-3β (Cell Signaling Technology), anti–phospho–GSK-3β (phosphorylated at Ser⁹; Cell Signaling Technology), anti-NLRP3 (AdipoGen), anti–caspase 1-p20 (AdipoGen) and anti-GAPDH antibodies (Santa Cruz Biotechnology). After washing, blots were incubated with their corresponding secondary antibodies. The signals on the membranes were detected via enhanced chemiluminescence analysis (Cell Signaling Technology).

Zhao J., Wang H., Huang Y., Zhang H., Wang S., Gaskin F., Yang N, & Fu S.M. (2015). Lupus Nephritis: Glycogen Synthase Kinase 3β Promotion of Renal Damage Through Activation of the NLRP3 Inflammasome in Lupus-Prone Mice. Arthritis & rheumatology (Hoboken, N.J.), 67(4), 1036-1044.

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Western Blot Analysis of Cell Signaling

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Nitrocellulose membranes containing the transferred proteins were blocked in Intercept Blocking Buffer (LI-COR Bioscience) for 1-hour at room temperature. Membranes were probed with primary antibodies, including mouse anti-mTOR (1:1000; Cat# 4517), mouse anti-Akt (1:1000; Cat# 2920), mouse anti-GSK3β (1:1000; Cat# 9832), mouse anti-ERK (1:1000; Cat# 9107), rabbit anti-phospho mTOR (1:1000; Cat# 5536 S), rabbit anti-phospho Akt (1:1000; Cat# 4060 S), rabbit anti-phospho GSK3β (1:1000; Cat# 5558), rabbit anti-phospho ERK (1:1000; Cat# 4376) (all antibodies were purchased from Cell Signaling Technologies) diluted in Intercept Blocking Buffer + 0.2% Tween-20 and incubated overnight at 4 °C, before being exposed to secondary antibody (IRDye^® 680RD Donkey anti-Mouse IgG and IRDye^® 800CW Donkey anti-Rabbit IgG) (LI-COR Biosciences) for 1-hour at room temperature in the dark.
Membranes were scanned on the LI-COR Bioscience Odyssey CLx imaging system and imaged using LI-COR Image Studio software version 2.1.10. All densitometry analyses were performed using Image Studio Light version 5. The region of interest encircling each band was defined automatically. All bands at the correct molecular weight were analyzed as the signal for that target protein. Values for each protein were normalized to loading control β-tubulin (Abcam).

Price J.B., Yates C.G., Morath B.A., Van De Wakker S.K., Yates N.J., Butters K., Frye M.A., McGee S.L, & Tye S.J. (2021). Lithium augmentation of ketamine increases insulin signaling and antidepressant-like active stress coping in a rodent model of treatment-resistant depression. Translational Psychiatry, 11, 598.

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Antibody Identification and Utilization

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For antibodies, rabbit anti-DISC1 (Millipore, ABN-425) was purchased from Millipore; rabbit anti-BMAL1 (Abcam, ab93806) from Abcam; mouse anti-alpha-tubulin (Proteintech, 66031-1-lg) from Proteintech; rabbit anti-HA (Bethyl, A190-108A) from Bethyl; rabbit anti-GFP (Invitrogen, A11122) from Invitrogen; rabbit anti-Flag (ThermoFisher, PA1-984B) from ThermoFisher; mouse anti-Flag (Sigma, F1804) from Sigma; mouse anti-c-Myc (Santa Cruz, sc-40) from Santa Cruz; GSK3β p-Y216 (Invitrogen, 44-604G) from Invitrogen; mouse anti-GSK3β (Cell Signaling, 9832S) from Cell Signaling. Antibodies were utilized as the first antibody of western blot or precipitating antibody of immunoprecipitation.

Lee S.B., Park J., Kwak Y., Park Y.U., Nhung T.T., Suh B.K., Woo Y., Suh Y., Cho E., Cho S, & Park S.K. (2021). Disrupted-in-schizophrenia 1 enhances the quality of circadian rhythm by stabilizing BMAL1. Translational Psychiatry, 11, 110.

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Cardioprotective Effects of DEX

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DEX was purchased from Jiangsu Nhwa Pharmaceutical Corporation Ltd. (Jiangsu, China). Wortmannin (Wort) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Rat CK-MB isoenzyme enzyme linked-immunosorbent assay (ELISA) kit (cat. no. E006), LDH assay kit cat. no. A020-2), cTnI ELISA kit (cat. no. H149-2), SOD assay kit (cat. no. A001-3) and MDA assay kit (cat. no. A003-1) were obtained from Jiancheng Institute of Biotechnology (Nanjing, China). TRIzol^® reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Bcl-2, Bax and β-actin primers were acquired from Sangon Biotech Co., Ltd. (Shanghai, China). The following primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers MA, USA): Mouse anti-GSK-3β (1:2,000 dilution; cat. no. BF0695), rabbit polyclonal anti-p-GSK-3β (1:2,000 dilution; cat. no. AF2016), rabbit polyclonal anti-Akt (1:1,000 dilution; cat. no. 9272S), rabbit polyclonal anti-p-Akt (1:1,000 dilution; cat. no. 4060S) and rabbit polyclonal anti-cleaved caspase-3 (1:2,000 dilution; cat. no. 9661S). Horseradish peroxidase (HRP)-linked anti-mouse immunoglobulin (IgG) (1:10,000 dilution; cat. no. BA1050), HRP-linked anti-rabbit IgG (1:10,000 dilution; cat. no. BA1054) and anti-β-actin (1:1,000 dilution; cat. no. BM0627) were acquired from Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China).

CHENG X.Y., GU X.Y., GAO Q., ZONG Q.F., LI X.H, & ZHANG Y. (2016). Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury. Molecular Medicine Reports, 14(1), 797-803.

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Quantifying GSK-3β and NF-κBp65 Signaling in Rat Brain

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To determine the levels of GSK-3β, phosphorylated GSK-3β (p-GSK-3β), NF-κBp65 and phosphorylated NF-κBp65 (p-NF-κBp65) in the cortex and hippocampus, 50 mg of rat brain tissue was lysed in 500 μL of lysis buffer and incubated with rabbit anti-p-GSK-3β (1:1,000), mouse anti-GSK-3β (1:1,000), rabbit anti-p-NF-κBp65 (1:500) or mouse anti-NF-κBp65 (1:500) (all from Cell Signaling Technology).

Liu H., Ou S., Xiao X., Zhu Y, & Zhou S. (2015). Diabetes Worsens Ischemia-Reperfusion Brain Injury in Rats Through GSK-3β. The American Journal of the Medical Sciences, 350(3), 204-211.

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Immunocytochemistry and Western Blotting of Alzheimer's Markers

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The primary antibodies used for immunocytochemistry in this study were rabbit anti-Aβ42 (1:35, Millipore), mouse anti-NES (nestin) (1:500, R&D), rabbit anti-OCT4 (1:500, Abcam), mouse anti-SYP (synaptophysin) (1:500, Abcam), mouse anti-TRA-1-81 (1:250, BD Pharmingen), and rabbit anti-TUBB3 (tubulin β3 class III) (1:1000, Covance). The secondary antibodies included Alexa594-conjugated donkey anti-rabbit IgG (1:200, Invitrogen), Alexa488-conjugated donkey anti-goat IgG (1:200, Invitrogen), and Alexa488-conjugated donkey anti-mouse IgG (1:200, Invitrogen). The antibodies used for western blotting were mouse anti-GSK3β (glycogen synthase kinase 3β) (1:500, Cell Signaling Technology), mouse anti-phospho-GSK3β (Ser9) (1:500, Cell Signaling Technology), mouse anti-tau (1:500, Abcam), mouse anti-phospho-tau (Ser396) (1:1000, sigma), mouse anti-phospho-tau (Thr181) (1:500, Cell Signaling Technology), and mouse anti-TUBB3 (1:10000, Biolegend). The secondary antibodies used included goat anti-mouse IgG-HRP (horseradish peroxidase) (1:20000, GE Healthcare), goat anti-rabbit IgG-HRP (1:20000, GE Healthcare), and donkey anti-goat IgG-HRP (1:2500, Santa Cruz). The immune complexes were detected using an enhanced chemiluminescent substrate (ECL, Biotools).

Chang K.H., Lee-Chen G.J., Huang C.C., Lin J.L., Chen Y.J., Wei P.C., Lo Y.S., Yao C.F., Kuo M.W, & Chen C.M. (2018). Modeling Alzheimer’s Disease by Induced Pluripotent Stem Cells Carrying APP D678H Mutation. Molecular Neurobiology, 56(6), 3972-3983.

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