- MA5-24727 - Invitrogen Antibodies USP14 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Anti-USP14 Monoclonal Antibody (889CT6.2.1) (Product # MA5-24727) was achieved by transfecting HEK-293 with USP14 specific siRNAs (Silencer® select Product # S17360, S17358). Western blot analysis (Fig. a) was performed using Whole cell extracts from the USP14 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with USP14 Monoclonal Antibody (889CT6.2.1) (Product # MA5-24727, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000 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 USP14.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-USP14 Monoclonal Antibody (889CT6.2.1) (Product # MA5-24727) and a 56 kDa band corresponding to USP14 was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of NIH:OVCAR-3 (Lane 1), BeWo (Lane 2), HEK-293 (Lane 3), HCT 116 (Lane 4), HeLa (Lane 5), A-431 (Lane 6), THP-1 (Lane 7) and Neuro-2a (Lane 8). Tissue extracts of Mouse Brain (Lane 9) and Rat Brain (Lane 10) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX), 12 well. 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-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of USP14 in k562, Jurkat cell line, mouse brain and rat brain lysate using 35 µg of sample per lane. The membrane was probed with USP14 monoclonal antibody (Product # MA5-24727).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Anti-USP14 Monoclonal Antibody (889CT6.2.1) (Product # MA5-24727) was performed using 70% confluent log phase A549 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 45 minutes at room temperature. The cells were labeled with USP14 Monoclonal Antibody (889CT6.2.1) (Product # MA5-24727) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). 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 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41).

MA5-24727