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Figure 6


Fig. 6. Turnover of wild-type and R664X AE1 proteins in HEK293 cells. (A) Cell-surface proteins in HEK293 cells transiently transfected with wild-type (WT) and R664X (RX) AE1 were biotinylated and polypeptides in total cell lysate (T) were separated into bound (B) and unbound (U) fractions on NutrAvidin beads, followed by detection of AE1 proteins by immunoblotting. Each lane contained proteins from the equivalent volume of cell lysate. (B) HEK293 cells transiently transfected with wild type (WT), R664X (RX), wild type and GPC (WT/GPC), or wild-type and R664X AE1 (WT/RX) were pulse-labeled for 20 minutes and chased for the indicated periods followed by immunoprecipitation of AE1 as described for Fig. 1. Lanes 10+ contained immunoprecipitates from the cells chased for 10 hours in the presence of 10 µM lactacystin. The lower panel shows wild-type and R664X proteins remaining after the chase in the presence ({blacksquare}) or absence ({circ}) of lactacystin. Data are the mean values of duplicate samples. Pulse-labeled wild-type and R664X AE1 in WT/GPC and WT/RX HEK293 cells showed stability similar to that observed in HEK293 cells transfected with wild type or R664X alone (data not shown).





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