Figure 1

A. Expression of OPN is down-regulated by 95% in the 435/BRMS1 cells. Serum-free conditioned medium from equal numbers of pcDNA transfected MDA-MB-435 (V) and 901-BRMS1-transfected 435 cells, 435/BRMS1 (BRMS1) was resolved using a 12.5% SDS-PAGE, transferred to a PVDF membrane and probed with the anti-human OPN monoclonal antibody [30]. The bar graph depicts the percent change in OPN expression based on densitometric analysis of the immunoblot. To confirm the BRMS1 expression in these cells, the cells were lysed in NP-40 lysis buffer and 30 ug of protein was resolved using a 12.5% SDS-PAGE, transferred to a PVDF membrane and probed with the anti-901 monoclonal antibody (for BRMS1 epitope). The membrane was reprobed for levels of β-actin to confirm equal loading. B. BRMS1 suppresses activity of the human OPN promoter. COS-7 cells were co-transfected with pGL3-OPN [28] and pCMV-myc or pCMV-myc-BRMS1 using Lipofectamine 2000 (Invitrogen). Luciferase activity was normalized to the total protein concentration. Data is expressed as Relative luciferase activity, where control is 100%. The data represents five independent experiments in triplicate. * indicates significant suppression (p < 0.05).