Figure 2

Immunoprecipitation of target antigens and confirmation of CD73 as a target. Immunoprecipitation (A) of CD73 (i) and integrin α3 (ii) from MDA-MB-231 cell lysate. MDA-MB-231 cell lysate was incubated in the presence of antibody, coupled to Sepharose. The Sepharose was washed with D-PBS prior to bound antigens being eluted using 0.1 M glycine pH 2.7 (e1 and e2). All analysis was performed by silver stained SDS-PAGE. Confirmation of target antigen for the anti-CD73 antibody was performed by siRNA knockdown of CD73 on MDA-MB-231 cells (B). (i) Flow cytometry was used to detect CD73 expression 48 hours post-transfection. (ii) CD73 protein knockdown in response to CD73 siRNA was determined by western blot analysis 48 hours post-transfection. Actin was used as a loading control. Binding to other triple negative breast cancer cell lines, by the anti-CD73 antibody, was confirmed by FACS (C). (i) Binding to SUM159 and (ii) binding to BT-549.