- PA5-17634 - Invitrogen Antibodies MAPK9 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of JNK2 was achieved by transfecting HeLa with JNK2 specific siRNAs (Silencer® select Product # s11159). Western blot analysis (Fig. a) was performed using whole cell extracts from the JNK2 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with JNK2 Polyclonal Antibody (Product # PA5-17634, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25ug/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 JNK2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-JNK2 Polyclonal Antibody (Product # PA5-17634) and a 50 kDa band corresponding to JNK2 was observed in the cell lines and tissue tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), NIH/3T3 (Lane 2), PC-12 (Lane 3), THP-1 (Lane 4), Mouse brain (Lane 5) in Fig. a and HL-60 (Lane 1), Raji (Lane 2) in Fig. b were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of JNK2 was performed using 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with JNK2 Polyclonal Antibody (Product # PA5-17634) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant 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 staining in nucleus and cytoplasm. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-17634