Figure 4.

Effect of DTT on proPC2 and 7B2 maturation and binding. AtT-20 cells that express 7B2 and PC2 were labeled for 10 min and chased for the indicated time periods. Cells treated with 5 mM DTT were in the continuous presence of the drug during the pulse and the chase. (a) DTT prevents proPC2 processing and secretion. Cells were extracted at the end of the pulse and after a 2-h chase. PC2 was immunoprecipitated from the cell extracts (Cells) and from the media (M) under denaturing conditions. (b) DTT does not affect 7B2 processing and secretion. Cells were extracted at the end of the pulse and after a 30-min chase. 7B2 was immunoprecipitated from the cell extracts and from the media under native conditions. (c) DTT reversibly prevents 7B2 binding to proPC2. Cells were first chased in the presence or absence of DTT for 30 min. Cells that had been incubated in the presence of DTT were then washed twice with PBS and further chased for 30 min in the presence or absence of DTT. 7B2 was immunoprecipitated under native conditions.