Figure 1.
The PC1/3N222D mutation affects insulin biosynthesis in vivo. A, Schematic showing proinsulin processing. Proinsulin intermediate 1 is cleaved at the C-A junction, and proinsulin intermediate 2 is cleaved at the B-C junction. B, One hundred pancreatic islets were preincubated in DMEM containing 25.5mM glucose for 30 minutes. After being briefly washed twice with Cys/Met-deficient DMEM medium, the islets were pulse labeled with 35S-Cys/Met in the same medium for 20 minutes. The labeled islets were split in half: 1 was lysed immediately, and the other was chased in RPMI 1640 for 4 hours. Newly synthesized proinsulin, conversion intermediates, and insulin were recovered by immunoprecipitation with antiinsulin and analyzed by Tris-tricine-urea-SDS-PAGE under nonreducing conditions. C, Islets were labeled for 20 minutes and chased in RPMI 1640 medium for times indicated. The islets were lysed. The lysates and chase media were combined and immunoprecipitated with antiinsulin followed by Tris-tricine-urea-SDS-PAGE under reducing conditions.