- MA3-038 - Invitrogen Antibodies GSK3B antibody

Submitted references Piwi reduction in the aged niche eliminates germline stem cells via Toll-GSK3 signaling.

Lin KY, Wang WD, Lin CH, Rastegari E, Su YH, Chang YT, Liao YF, Chang YC, Pi H, Yu BY, Chen SH, Lin CY, Lu MY, Su TY, Tzou FY, Chan CC, Hsu HJ
Nature communications 2020 Jun 19;11(1):3147

Sevoflurane inhibits the proliferation and invasion of hepatocellular carcinoma cells through regulating the PTEN/Akt/GSK‑3β/β‑catenin signaling pathway by downregulating miR‑25‑3p.

Cao Y, Lv W, Ding W, Li J
International journal of molecular medicine 2020 Jul;46(1):97-106

Translation of the circular RNA circβ-catenin promotes liver cancer cell growth through activation of the Wnt pathway.

Liang WC, Wong CW, Liang PP, Shi M, Cao Y, Rao ST, Tsui SK, Waye MM, Zhang Q, Fu WM, Zhang JF
Genome biology 2019 Apr 26;20(1):84

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of GSK-3 was performed by loading 20 µg of the indicated whole cell lysates and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a Novex 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BOX ). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% Milk in TBST for 1 hour at room temperature. GSK-3 (alpha and beta isoforms) was detected at ~51 kD and ~47 kD using a GSK-3 monoclonal antibody (Product # MA3-038) at a dilution of 1:1000 in 5% Milk in TBST overnight at 4C on a rocking platform, followed by a Goat anti-Mouse IgG Secondary Antibody, HRP conjugate (Product # 31430) at a dilution of 1:10,000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal Pico substrate (Product # 34078) and the myECL Imager (Product # 62236).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of GSK3 alpha/beta was achieved by transfecting MCF7 cells with GSK3 alpha/beta specific siRNAs (Silencer® select Product # s6236, s6240). Western blot analysis (Fig. a) was performed using whole cell extracts (1% SDS) from the GSK3 alpha/beta knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with GSK3 alpha/beta Monoclonal Antibody (Product # MA3-038, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Decrease in signal upon siRNA mediated knockdown confirms that antibody is specific to GSK3 alpha/beta.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MCF7 (Lane 1) and MDA-MB-231 (Lane 2), HeLa (Lane 3), Neuro-2a (Lane 4), U-87 MG (Lane 5), A-431 (Lane 6), COS-7 (Lane 7) and NIH/3T3 (Lane 8). The blot was probed with Anti-GSK3 alpha/beta Mouse Monoclonal Antibody (Product # MA3-038, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 51 kDa and 47 kDa band corresponding to GSK3 alpha and beta was detected across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of GSK-3 (green) in 3T3 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 30 minutes at room temperature. Cells were stained with a GSK-3 monoclonal antibody (Product # MA3-038) at a dilution of 1:1000 in staining buffer for 1 hour at room temperature, and then incubated with a Goat anti-Mouse IgG Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Product # A28175) at a dilution of 1:1000 for 1 hour at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of GSK-3 was done on HeLa cells. Cells were fixed, permeabilized and stained with a GSK-3 mouse monoclonal antibody (Product # MA3-038, red histogram) at a dilution of 1:100. After incubation for 1 hour on ice, the cells were labeled with DyLight 680 Goat Anti-Mouse Secondary Antibody (Product # 35518) at a dilution of 1:50 for 1 hour on ice. A representative 10,000 cells were acquired and analyzed for each sample. The black histogram represents unstained control cells and the blue histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of GSK3 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of GSK3 monoclonal antibody (Product # MA3-038) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a GSK3 monoclonal antibody (Product # MA3-038) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of GSK3 was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of GSK3 monoclonal antibody (Product # MA3-038) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti-mouse coated Dynabeads (Product # 11202D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a GSK3 monoclonal antibody (Product # MA3-038) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western Blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Sevo blocks the PTEN/Akt/GSK-3beta/beta-catenin signaling pathway by modulating the expression of miR-25-3p in HCC cells. HCCLM3 and Huh7 cells were transfected with miR-25-3p mimics or miR-25-3p inhibitor for 24 h followed by Sevo treatment for 6 h. (A) Protein levels of p-Akt, Akt, p-GSK-3beta, GSK-3beta, beta-catenin, c-Myc and MMP9 were measured using western blotting. beta-actin served as the loading control. (B) The bands were semi quantitatively analyzed using ImageJ and normalized to beta-actin density. Data are presented as the mean +- standard deviation. (n=3) of three representative experiments. * P

MA3-038

Antibody data