All lanes : Anti-Fos B antibody [EPR23489-90] (ab252237) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) serum starved overnight, whole cell lysate
Lane 2 : HeLa serum starved overnight, then treated with 200 nM 12-o-tetradecanoyl phorbol 13-acetate (TPA, ab120297) for 4 hours, whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 36 kDa
Observed band size: 37/48 kDa why is the actual band size different from the predicted?

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

The band around 37kDa is caused by splicing isoform.

The expression profile observed is consistent with what has been described in the literature (PMID: 9204924).

Exposure time: 37 seconds.

Fos B was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) serum starved overnight, then treated with 200 nM TPA for 4 hours, whole cell lysate 10 µg with ab252237 at 1/30 dilution (2µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab252237 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HeLa serum starved overnight, then treated with 200 nM TPA for 4 hours, whole cell lysate 10 µg.

Lane 2: ab252237 IP in HeLa serum starved overnight, then treated with 200 nM TPA for 4 hours, whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252237 in HeLa serum starved overnight, then treated with 200 nM TPA for 4 hours, whole cell lysate.

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

Exposure time: 5 seconds.

The band around 37kDa is caused by splicing isoform.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) was serum starved for 16 hours, then treated with 200nM 12-O-Tetradecanoylphorbol-13-acetate (TPA) for 4 h (Red) / Untreated control (Green) cells labeling Fos B with ab252237 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Fos B with ab252237 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cells treated with starvation 16 hours, then 12-O-Tetradecanoylphorbol-13-acetate (200nM) for 4 hours. 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: ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling Fos B with ab252237 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive nuclear staining in cancer cells of human breast cancer is observed (PMID: 12602926). The section was incubated with ab252237 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human hippocampus tissue labeling Fos B with ab252237 at 1/500 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive nuclear staining in neurons of human hippocampus is observed (PMID: 29805976). The section was incubated with ab252237 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.