- PA5-17476 - Invitrogen Antibodies NEDD8 antibody

Submitted references Effects of the NEDD8-Activating Enzyme Inhibitor MLN4924 on Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.

Chang PJ, Chen LW, Chen LY, Hung CH, Shih YJ, Wang SS
Journal of virology 2017 Oct 1;91(19)

Effects of Exogenous NUB1 Expression in the Striatum of HDQ175/Q7 Mice.

Vodicka P, Chase K, Iuliano M, Valentine DT, Sapp E, Lu B, Kegel-Gleason KB, Sena-Esteves M, Aronin N, DiFiglia M
Journal of Huntington's disease 2016 Jun 13;5(2):163-74

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Nedd8 was performed by loading whole cell lysate in 1X LDS sample buffer with 2-ME from 5 x 10^5 mouse transformed pre-B cells. Samples were loaded onto a 4-12 % Bis-Tris polyacrylamide gel (Product # NP0336BOX). Proteins were transferred to nitrocellulose membrane with wet/tank transfer. Membrane was blocked in 5% milk/TBST. The membrane was probe with a polyclonal anti-Nedd8 antibody (Product # PA5-17476) at a dilution of 1:500 in 5% milk/TBST at 4oC overnight, followed by a secondary antibody HRP-anti-rabbit at a dilution of 1:10000 at room temperature for 1 hour. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # PI-32209). Data courtesy of Thermo Scientific KOL Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NEDD8 recombinant protein (5 ng) using NEDD8 polyclonal antibody (Product # PA5-17476).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of NEDD8 in lysates from HeLa and Raji cells using NEDD8 polyclonal antibody (Product # PA5-17476).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NEDD8 was achieved by transfecting HeLa with NEDD8 specific siRNAs (Silencer® select Product # s226684, s9424). Western blot analysis (Fig. a) was performed using whole cell extracts from theNEDD8 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NEDD8 Polyclonal Antibody (Product # MA5-17476, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 NEDD8.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using anti-NEDD8 Polyclonal Antibody (Product # PA5-17476) and a 9 kDa band corresponding to NEDD8 was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), Jurkat (Lane 3), PC-12 (Lane 4), THP-1 (Lane 5), K-562 (Lane 6), HL-60 (Lane 7), Raw 264.7 (Lane 8), tissue extracts of Rat testis (Lane 9), Mouse brain (Lane 10) and Mouse colon (Lane 11) were electrophoresed Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). 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, 1:4000 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)
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Experimental details: Immunofluorescence analysis of NEDD8 was performed using 70% confluent log phase HeLa 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 NEDD8 Polyclonal Antibody (Product # PA5-17476) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled 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 localization to the cytoplasm and nucleus. 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: Immunohistochemical analysis of NEDD8 in paraffin-embedded human colon carcinoma using a NEDD8 polyclonal antibody (Product # PA5-17476) showing cytoplasmic and membrane localization.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIG 6 LANA is naturally modified with NEDD8 in cells. (A) Neddylation of LANA in 293T cells. 293T cells were transfected with the indicated plasmids expressing HA-NEDD8 or F-LANA and left untreated or treated with MLN4924 (2.0 muM) for 24 h. Cell samples were then subjected to immunoprecipitation (IP) and immunoblot (IB) analysis. (B) Neddylation of LANA in 293T(BAC16) cells. 293T(BAC16) cells were transfected with the expression plasmid for HA-NEDD8 and cultured in medium with or without MLN4924 (2.0 muM) for 24 h. Cell lysates were immunoprecipitated using anti-LANA antibody, and the resultant immunoprecipitates were analyzed by immunoblotting using antibodies against LANA, HA, and NEDD8. (C) Neddylation of LANA in BCBL1 cells. BJAB and BCBL1 cells were left untreated or treated with MLN4924 (2.0 muM) for 24 h. After cell lysates were immunoprecipitated with anti-LANA antibody, the immunoprecipitated proteins were probed with anti-LANA and anti-NEDD8 antibodies. Arrows indicate the positions of the neddylated LANA.

PA5-17476

Antibody data