Figure 6

(A) RNAi mediated reduction of Sec5 and Sec6 expression. R3327-5′A were transfected with either nothing (“mock”) or with siRNAs targeting Sec5, Sec6 or a control non-targeting siRNA (“control”), as described in Materials & Methods. 60 hr post-transfection, cells were lysed and lysates were analyzed by SDS-PAGE and immunoblotting for Sec5, Sec6 and β-PIX. Protein levels were quantified using a Molecular Dynamics Typhoon phosphorimager. (B) Metabolic pulse-chase and surface biotinylation analysis of α5 integrin trafficking in prostate tumor cells. Delivery of newly synthesized α5-GFP to the surface of R3327-5′A cells was assessed as described in Materials and Methods. Experiments were performed twice, each time with triplicate wells of cells. Relative surface delivery was defined as the mean signal obtained from three replicate biotinylated α5-GFP bands, normalized to the mean of the total α5-GFP recovered in the initial immunoprecipitates.