- PA5-30301 - Invitrogen Antibodies USP14 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of USP14 using 50 µg of mouse brain lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a USP14 polyclonal antibody (Product # PA5-30301) at a dilution of 1:5000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of USP14 was performed by separating 50 µg of various tissue extracts by 10% SDS-PAGE. Proteins were transferred to a membrane and probed with a USP14 Polyclonal Antibody (Product # PA5-30301) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using USP14 Polyclonal Antibody (Product # PA5-30301). Sample (30 µg of whole cell lysate). Lane A: A431 . 7.5% SDS PAGE. USP14 Polyclonal Antibody (Product # PA5-30301) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Anti-USP14 Polyclonal Antibody (Product # PA5-30301) 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 Polyclonal Antibody (Product # PA5-30301, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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)
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Experimental details: Western blot was performed using Anti-USP14 Polyclonal Antibody (Product # PA5-30301) and a 56 kDa band along with unncharacterized bands seen approximately at 35 kDa and 110 kDa corresponding to USP14 was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), NIH:OVCAR-3 (Lane 2), BeWo (Lane 3), HEK-293 (Lane 4), HCT 116 (Lane 5), HeLa (Lane 6), A-431 (Lane 7), THP-1 (Lane 8) and Neuro-2a (Lane 9), tissue extracts of Mouse Brain (Lane 10) and Rat Brain (Lane 11) 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-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of USP14 was performed in DIV14 rat E18 primary cortical neurons fixed in 4% paraformaldehyde at RT for 15 min. Green: USP14 Polyclonal Antibody (Product # PA5-30301) diluted at 1:500. Red: beta Tubulin 3/ Tuj1, stained by beta Tubulin 3/ Tuj1 antibody. Blue: Fluoroshield with DAPI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Anti-USP14 Polyclonal Antibody (Product # PA5-30301) 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 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with USP14 Polyclonal Antibody (Product # PA5-30301) 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 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 Cell Insight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al./Methods 115 (2017) 28–41).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry (Frozen) analysis of USP14 was performed in frozen-sectioned adult mouse cerebellum tissue using USP14 Polyclonal Antibody (Product # PA5-30301) at a dilution of 1:250 (Green). Red: beta Tubulin 3/ TUJ1, stained by beta Tubulin 3/ Tuj1 antibody diluted at 1:500. Blue: Fluoroshield with DAPI.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded human normal liver, using USP14 (Product # PA5-30301) antibody at 1:250 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-30301