Anti-NUR77 antibody [EPR29846-533]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Parental HeLa (human cervical adenocarcinoma epithelial cell) and NR4A1 KO HeLa (NR4A1 knockout human cervical adenocarcinoma epithelial cell) cells labelling NUR77 with ab324680 at 1/50 (10.08 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in wild-type HeLa cells(shown in green) with no staining in NR4A1 knockout HeLa cells. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• IP

Lab

Immunoprecipitation - Anti-NUR77 antibody [EPR29846-533] (AB324680)

NUR77 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab324680 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324680 at 1/324680 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab324680 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab324680 in HeLa whole cell lysate

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

Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-NUR77 antibody [EPR29846-533] (ab324680) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab324680 at 1/30 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324680 in HeLa whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 75 kDa

false

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling NUR77 with ab324680 at 1/500 (1.008 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse lung. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling NUR77 with ab324680 at 1/500 (1.008 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse cerebrum. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-12 (rat adrenal gland pheochromocytoma cell) and PC-12 treated with MG-132 (10uM) for 6hours cells labelling NUR77 with ab324680 at 1/50 (10.08 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased nuclear staining in PC-12 cell line (shown in green) treated with MG-132 (10uM) for 6h. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) and NIH/3T3 treated with MG-132 (10uM) for 6hours cells labelling NUR77 with ab324680 at 1/50 (10.08 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing increased nuclear staining in NIH/3T3 cell line (shown in green) treated with MG-132 (10uM) for 6h. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunohistochemical analysis of paraffin-embedded Mouse T cell lymphoma tissue labeling NUR77 with ab324680 at 1/500 (1.008 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse T cell lymphoma. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling NUR77 with ab324680 at 1/500 (1.008 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat lung. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NUR77 antibody [EPR29846-533] (AB324680)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling NUR77 with ab324680 at 1/500 (1.008 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat cerebrum. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Lab

Immunoprecipitation - Anti-NUR77 antibody [EPR29846-533] (AB324680)

NUR77 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab324680 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab324680 at 1/324680 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 2 : ab324680 at 1/30 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab324680 in NIH/3T3 whole cell lysate

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

Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-NUR77 antibody [EPR29846-533] (ab324680) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

ab324680 at 1/30 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab324680 in NIH/3T3 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-NUR77 antibody [EPR29846-533] (AB324680)

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

Performed under reducing conditions.

In Western blot, ab324680 was shown to bind specifically to NUR77. Target of interest was observed at 65-75 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in NR4A1 knockout cell line (lane 2) (lane 2, knockout cell line ab264021 / knockout cell lysate ab264509).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-NUR77 antibody [EPR29846-533] (ab324680) at 1/1000 dilution

Lane 1:

Wild type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

NR4A1 knockout HeLa whole cell lysate at 50 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 1/100000 dilution

Observed band size: 65-75 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-NUR77 antibody [EPR29846-533] (AB324680)

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 29669342).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 3 seconds. Lanes 3-6 : 15 seconds.

All lanes:

Western blot - Anti-NUR77 antibody [EPR29846-533] (ab324680) at 1/1000 dilution

Lane 1:

Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 50 µg

Lane 2:

NIH/3T3 treated with 10 µM MG-132 for 6 hours, whole cell lysate at 50 µg

Lane 3:

Untreated PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 50 µg

Lane 4:

PC-12 treated with 10 µM MG-132 for 6 hours, whole cell lysate at 50 µg

Lane 5:

Untreated 3T3-L1 (mouse embryonic fibroblast) whole cell lysate at 50 µg

Lane 6:

3T3-L1 treated with 100 nM TPA for 2 hours, whole cell lysate at 50 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 1/100000 dilution

Observed band size: 65-75 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-NUR77 antibody [EPR29846-533] (AB324680)

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 29669342).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-NUR77 antibody [EPR29846-533] (ab324680) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 50 µg

Lane 2:

Rat skeletal muscle tissue lysate at 50 µg

Lane 3:

Mouse Ovary tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 65-75 kDa,36 kDa

false

Exposure time: 180s