All lanes : Anti-SMARCD1 antibody [EPR23170-71] (ab245222) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : SMARCD1 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 58 kDa
Observed band size: 58 kDa

Lanes 1-4: Merged signal (red and green). Green - ab245222 observed at 58 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab245222 Anti-SMARCD1 antibody [EPR23170-71] was shown to specifically react with SMARCD1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266458 (knockout cell lysate ab259143) was used. Wild-type and SMARCD1 knockout samples were subjected to SDS-PAGE. ab245222 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling SMARCD1 with ab245222 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in Neuro-2a cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

SMARCD1 was immunoprecipitated from 0.35 mg of NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate with ab245222 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab245222 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: NIH/3T3 whole cell lysate 10 (Input).
Lane 2: ab245222 IP in NIH/3T3 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab245222 in NIH/3T3 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling SMARCD1 with ab245222 at 1/500 dilution (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.

All lanes : Anti-SMARCD1 antibody [EPR23170-71] (ab245222) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 2 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg
Lane 4 : Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 58 kDa
Observed band size: 58 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

SMARCD1 was immunoprecipitated from 0.35 mg of HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab245222 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab245222 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 (Input). 
Lane 2: ab245222 IP in HeLa whole cell lysate. 
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab245222 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling SMARCD1 with ab245222 at 1/500 dilution (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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling SMARCD1 with ab245222 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.