All lanes : VIME Mouse mAb at 1/2000 dilution Lane 1 : Jurkat cells membrane fraction Lane 2 : THP1 cells membrane fraction Lysates/proteins at 20 μg per lane. Secondary Goat Anti-Mouse IgG-HRP, 5% Skim milk conjugated at 1/10000 dilution Predicted band size : 54 KDa Observed band size : 54 KDa Blocking/Dilution buffer : 1× PBS.

VIME was immunoprecipitated from 1mg of Jurkat cells membrane fraction, blotted with M25003 of 10μg. Western blot was performed from the immunoprecipitate using M25003 at 1/2000 dilution. Anti-Mouse-IgG(HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/10000 dilution. Lane 1: Jurkat cells membrane fraction. Lane2:IP product of Jurkat cells membrane fraction. Blocking and dilution buffer and concentration:5% milk/TBST.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling VIME with M25003 at 1/200 dilution (red) compared with a mouse monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody,blue). Goat anti-mouse IgG (FITC) at 1/300 dilution was used as the secondary antibody.