- 702286 - Invitrogen Antibodies TRAF6 antibody

Submitted references TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation.

Pradhan P, Toy R, Jhita N, Atalis A, Pandey B, Beach A, Blanchard EL, Moore SG, Gaul DA, Santangelo PJ, Shayakhmetov DM, Roy K
Science advances 2021 Jan;7(3)

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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 HeLa (Lane 1), K-562 (Lane 2), HEK-293 (Lane 3), COS-7 (Lane 4), Jurkat (Lane 5), PC-3 (Lane 6), NIH/3T3 (Lane 7), MCF-7 (Lane 8) and PANC-1 (Lane 9). The blots were probed with Anti-TRAF6 Recombinant Rabbit Monoclonal Antibody (Product # 702286, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 70 kDa band corresponding to TRAF6 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. 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: Western blot was performed using Anti-TRAF6 Recombinant Rabbit Monoclonal Antibody (1H4L2) (Product # 702286) and a ~70 kDa band corresponding to TNF receptor-associated factor 6 was observed in Mouse Thymus and Mouse Lungs; and was absent in Mouse Liver. Whole cell extracts (30 µg lysate) of Mouse Thymus (Lane 1), Mouse Lung (Lane 2) and Mouse Liver (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescentdetection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of TNF receptor-associated factor 6 was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with TRAF6 Recombinant Rabbit Monoclonal Antibody (1H4L2) (Product # 702286) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Sustained IRF5 phosphorylation, higher IRF5 nuclear translocation, and elevated TRAF6 are responsible for synergistic IFN and IL-12p70 responses in BM-APCs. ( A ) Schematic showing TRAF6 and IRF5 signaling following TLR4 and TLR9 activation. ( B ) Levels of phosphorylated (Phos-tag) and total IRF5 [SDS-polyacrylamide gel electrophoresis (PAGE)] after 0.5, 1, 2, 4, 6, and 24 hours of BM-APCs treated with MPs with MPLA and/or CpG. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control, and treated IRF5 -/- BM-APCs were used as negative controls. ( C ) Total IRF5 levels in the nuclear and cytoplasmic fractions at 1, 2, 4, 6, 12, and 24 hours of BM-APC treated with MPs with MPLA and/or CpG. Ratio of nuclear-to-cytoplasmic IRF5 after 1, 2, 4, 6, 12, and 24 hours of treatment, where a higher ratio indicates a higher rate of nuclear translocation. Ratios were performed by Bio-Rad Image Lab software. ( D ) Kinetics of IFN-beta and IL-12p70 production by BM-APCs treated with PLPs. BM-APCs from this experiment were used for nuclear fractionation studies, and the results are shown in (C). ( E ) Levels of total TRAF6 (SDS-PAGE) after 0.5, 1, 2, 4, 6, and 24 hours of BM-APCs treated with MPs with MPLA and/or CpG. GAPDH was used as a loading control, and TRAF6 knocked down in BM-APCs was used as a negative control. On the right is the graphical representation of the GAPDH normalization of each blot. This was done with Bio-Rad Image Lab software.

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