All lanes : Anti-BRG1 antibody [EPR3912] (ab108318) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SMARCA4 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 185 kDa
Observed band size: 185 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab108318 observed at 185 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

 ab108318 was shown to react with SMARCA4 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255432 (knockout cell lysate ab263853) was used. Wild-type HEK-293T and SMARCA4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab108318 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 10000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder cancer tissue sections labeling BRG1 with purified ab108318 at 1/500 dilution (0.23 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : BRG1 knockout HAP1 cell lysate
Lane 3 : K562 cell lysate
Lane 4 : Molt-4 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 185 kDa
Additional bands at: 185 kDa. We are unsure as to the identity of these extra bands.

Lanes 1 - 4: Merged signal (red and green). Red - loading control, ab18058 (unpurified), observed at 124 kDa.

ab108318 was shown to specifically react with BRG1 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when BRG1 knockout samples were used. Wild-type and BRG1 knockout samples were subjected to SDS-PAGE, ab108318 and ab18058 (loading control to Vinculin) were diluted 1/1000 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BRG1 with purified ab108318 at 1/50 dilution (2.3 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BRG1 with purified ab108318 at 1/20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution (Purified)

Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 185 kDa
Observed band size: 185 kDa

Anti-BRG1 antibody [EPR3912] (ab108318) at 1/10000 dilution (Purified) + RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 185 kDa
Observed band size: 185 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat stomach tissue sections labeling BRG1 with purified ab108318 at 1/500 dilution (0.23 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse stomach tissue sections labeling BRG1 with purified ab108318 at 1/500 dilution (0.23 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

ab108318 (unpurified) staining BRG1 in wild-type HAP1 cells (top panel) and BRG1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab108318 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Formalin-fixed paraffin-embedded human colon tissue stained for BRG1 using ab108318 at 1/100 dilution in immunohistochemical analysis. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

All lanes : Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution (unpurified)

Lane 1 : K562 cell lysate
Lane 2 : Molt-4 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 185 kDa

ab108318 (unpurified) staining BRG1 in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab108318 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Formalin-fixed paraffin-embedded human Urinary bladder transitional carcinoma tissue.stained for BRG1 using ab108318 at 1/100 dilution in immunohistochemical analysis. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Formalin-fixed paraffin-embedded human Urinary Breast carcinoma tissue stained for BRG1 using ab108318 at 1/100 dilution in immunohistochemical analysis. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Formalin-fixed paraffin-embedded human Ovarian carcinoma tissue stained for BRG1 using ab108318 at 1/100 dilution in immunohistochemical analysis. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Formalin-fixed paraffin-embedded Normal human tonsil tissue stained for BRG1 using ab108318 at 1/100 dilution in immunohistochemical analysis. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.