All lanes : Anti-B7H4 antibody [EPR20236-9] (ab269455) at 1/1000 dilution

Lane 1 : SK-BR-3 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : T47D (human ductal breast epithelial tumor epithelial cell), whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 5 : Mouse uterus tissue 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: 31 kDa
Observed band size: 28,45,65 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 114 seconds.

B7H4 is N-glycosylated protein. The level of B7H4 glycosylation varies between different cells and tissues. The bands at 65kDa and 45kDa represent glycosylated B7H4. The band at 28kDa represents non-glycosylated B7H4. (PMID: 15878339).

Negative control: HT-29 and HeLa. (PMID: 15878339).

B7H4 was immunoprecipitated from 0.35 mg SK-BR-3 (Human breast adenocarcinoma epithelial cell) whole cell lysate with ab269455 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269455 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: SK-BR-3 whole cell lysate 10ug.

Lane 2: ab269455 IP in SK-BR-3 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269455 in SK-BR-3 whole cell lysate.

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

Exposure time: 30 seconds.

B7H4 is N-glycosylated protein. The level of B7H4 glycosylation varies between different cells and tissues. The band at 65kDa represents glycosylated B7H4. The band at 28kDa represents non-glycosylated B7H4 (PMID: 15878339).

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized HEK-293T (human embryonic kidney epithelial cell) transfected with myc-tagged B7-H4 expression vector cells labelling B7H4 with ab269455 at 1/60 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). PMID: 23722540 and PMID: 27632942 showed that cell surface expression of B7-H4 hard to detected in vitro cultured cell lines, and all the commercial B7H4 antibodies that recommend FC as an application use the overexpression system. Our RabMab also could recognise overexpressed B7H4.The overexpression plasmid contains the full length aa1-282 of human B7H4(Q7Z7D3) with HA tag in pcDNA3.1(+) vector.

Cells were surface stained with rabbit IgG (Left) or ab269455 (Right). Then fixed with 2% PFA followed by intracellularly stained with anti-HA tag conjugated to Alexa Fluor^® 647.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labelling B7H4 with ab269455 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and membranous staining in HEK-293T cell line transfected with a HA-tagged B7-H4 expression vector. Alexa Fluor^® 647 anti-HA.11 Epitope Tag Antibody was used at 200 dilution to detecte the overexpressed protein (Red). The nuclear counterstain was DAPI (Blue).

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