Fig. 7. GRIP domain overexpression affects proprotein convertase activity on a substrate protein, Pmel17. (a) Schematic diagram of Pmel17 primary structure and processed forms, as shown by Berson et al. (Berson et al., 2001). (b) HeLa cells were transiently transfected with expression vectors for Pmel17 (10 µg/10 cm dish) and either vector alone (lanes 1-4), C312 (lanes 5-8) or C312(Y2187A) (lanes 9-12) at 39 µg/10 cm dish. Two days post-transfection cells were harvested and pulse labeled with [³⁵S]methionine/cysteine for 30 minutes, and then chased for the indicated times. Pmel17 was immunoprecipitated from cell lysates at each time point, fractionated by SDS-PAGE, and visualized by phosphorimaging analysis. (c) Anti-HA immunoprecipitates from the same samples analyzed in the same way. Only the relevant portion of the gel encompassing C312 or C312(Y2187A) transgene products is shown.