Figure 1.

Secretion of newly synthesized proinsulin upon expression in AtT20 cells. The cells were pulse labeled for 30 min with ³⁵S-amino acids. During the indicated chase intervals (upper panels), sequential unstimulated (−) or stimulated (+) media and lysed cells were collected. In the lower panel, secretion into the medium beginning at 1.25 h of chase is shown, indicating that labeled proinsulin-derived peptides have already reached the stimulus-dependent secretory pathway by 1.25 h of chase. All samples were immunoprecipitated with anti-insulin antibody and analyzed by Tricine–urea–SDS-PAGE and fluorography. The positions of proinsulin and insulin are indicated; the band immediately above proinsulin probably represents the des-31,32 proinsulin conversion intermediate (Irminger et al., 1994). Results of a representative experiment (of four) are shown.