Anti-NUR77 antibody [EPR23982-85]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NUR77 antibody [EPR23982-85] (AB283264)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Jurkat (human T cell leukemia T lymphocyte) cells treated with 40nM TPA and 2 μM calcium ionophore for 4 hours (Red) or untreated control (Green) cells labelling NUR77 with ab283264 at 1/500 dilution (0.1 μg / Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-NUR77 antibody [EPR23982-85] (AB283264)

NUR77 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) cells treated with 10μM MG-132 for 6h whole cell lysate with ab283264 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283264 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa cells treated with 10μM MG-132 for 6h whole cell lysate 10 μg

Lane 2 : ab283264 IP in HeLa cells treated with 10μM MG-132 for 6h whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283264 in HeLa treated with 10μM MG-132 for 6h whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 32 seconds.

All lanes:

Immunoprecipitation - Anti-NUR77 antibody [EPR23982-85] (ab283264)

Predicted band size: 64 kDa

Observed band size: 38 kDa,70 kDa

false

• WB

Lab

Western blot - Anti-NUR77 antibody [EPR23982-85] (AB283264)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID : 30224829, 28111880, 29669342).

Exposure time : 15 seconds.

All lanes:

Western blot - Anti-NUR77 antibody [EPR23982-85] (ab283264) at 1/1000 dilution

Lane 1:

Untreated Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

Jurkat treated 40nM TPA and 2uM calcium ionophore A23187 for 4 hours whole cell lysate at 20 µg

Lane 3:

Jurkat treated 40nM TPA, 2uM calcium ionophore A23187 and 10uM MG-132 for 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 64 kDa

Observed band size: 32 kDa,38 kDa,60-80 kDa

false

• WB

Lab

Western blot - Anti-NUR77 antibody [EPR23982-85] (AB283264)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/molecular weight observed is consistent with what has been described in the literature (PMID : 30224829, 28111880, 29669342).

Exposure time : 15 seconds.

All lanes:

Western blot - Anti-NUR77 antibody [EPR23982-85] (ab283264) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa treated with 10uM MG-132 for 6h whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 64 kDa

Observed band size: 32 kDa,38 kDa,60-80 kDa

false

• WB

Lab

Western blot - Anti-NUR77 antibody [EPR23982-85] (AB283264)

False colour image of Western blot : Anti-NUR77 antibody [EPR23982-85] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab283264 was shown to bind specifically to NUR77. A band was observed at 62-70 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in NR4A1 knockout cell line ab264021 (knockout cell lysate ab264509). To generate this image, wild-type and NR4A1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-NUR77 antibody [EPR23982-85] (ab283264) at 1/1000 dilution

Lane 1:

Wild type HeLa treated MG132 (10 uM, 6 h) cell lysate at 20 µg

Lane 2:

NR4A1 knockout HeLa treated MG132 (10 uM, 6 h) cell lysate at 20 µg

Lane 2:

Western blot - Human NR4A1 (NUR77) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-nr4a1-nur77-knockout-hela-cell-line-ab264021'>ab264021</a>)

Lane 3:

Wild type HeLa vehicle control MG132 (0 uM, 6 h) cell lysate at 20 µg

Lane 4:

NR4A1 knockout HeLa vehicle control MG132 (0 uM, 6 h) cell lysate at 20 µg

Predicted band size: 64 kDa

Observed band size: 62-70 kDa

false