Lysosomal or proteasomal inhibition preserves 35S-methionine (Met) labeled nascent proinsulin in mouse islets/β-cells.
After 24-hour pre-experimental culture with the 5.5 mM (MIN6 β-cells) or 11 mM (islets) glucose concentration, MIN6 β-cells (A and B) or islets (C) were chased for the indicated times with/without chloroquine (100 µg/mL), lactacystin (10 µM), antimycin (10 µM), or brefeldin A (10 µg/mL) after a pulse for the indicated times with 35S-Met. Cellular proteins were then subjected to immunoprecipitation with insulin and C-peptide antisera. Equal amounts of individual immunoprecipitates were resolved by 10% tricine non-reduced and/or reduced SDS-PAGE for radiography. Relative levels of 35S-proinsulin monomers in (B) or (D) of individual immunoprecipitates (on the reduced gel) were shown in (A) and (C), respectively. The data in (A) or (C) were reported as mean ± SD. n = 3. *P<0.05; **P<0.01. % cont (Pro): percentage of proinsulin monomer level compared to the control.
