- 710132 - Invitrogen Antibodies GSK3B antibody

Wang R, Zhang Z, Kumar M, Xu G, Zhang M
Drug design, development and therapy 2019;13:501-512

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of GSK3 in whole cell extracts of HepG2 (lane 1) and NIH-3T3 (lane 2) using a GSK3 Recombinant Rabbit Polyclonal Antibody (Product # 710132) at a dilution of 1 µg/mL. Samples were detected using chemiluminescence (ECL). Results show a band at ~53kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts of MCF7 (Lane 1), MDA-MB-231 (Lane 2), HeLa (Lane 3), Neuro-2a (Lane 4), U-87 MG (Lane 5), A-431 (Lane 6) and NIH/3T3 (Lane 7). The blot was probed with Anti-GSK3 antibody (Product # 710132, 1µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 47 kDa band corresponding to GSK3B was observed across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of GSK-3-beta was performed by loading 30 µg of HeLa, HEK-293, Jurkat, MCF-7, DU 145, A431, COS-7, and COLO 205 cell lysates using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. GSK-3-beta was detected at ~47 kDa using GSK-3-beta Recombinant Rabbit Polyclonal Antibody (Product # 710132) at a 1:1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody (Product # G-21234) at a 1:5000 dilution and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of GSK3B was achieved by transfecting HeLa cells with GSK3B specific siRNAs (Silencer® select Product # s6240). Western blot analysis (Fig a) was performed using membrane enriched extracts from the GSK3B knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-GSK3B Recombinant Rabbit Polyclonal Antibody (Product # 710132, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/mL, 1:4000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to GSK3B.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of GSK3B was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR933148_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of GSK3B was performed by loading 30 µg of HeLa wild type (Lane 1) andHeLa GSK3B KO (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-GSK3B Recombinant Polyclonal Antibody (1HCLC) (Product # 710132, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5000 dilution) using the iBright™ FL 1500 (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to GSK3B.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of GSK3 in whole cell extracts of HepG2 (lane 1) and NIH-3T3 (lane 2) using a GSK3 Recombinant Rabbit Polyclonal Antibody (Product # 710132) at a dilution of 1 µg/mL. Samples were detected using chemiluminescence (ECL). Results show a band at ~53kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of GSK3B was performed using 70% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with GSK3 Rabbit Polyclonal Antibody (Product # 710132) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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