Validation of proteasomal or lysosomal roles in the disposal of proinsulin.

After 24-hour pre-experimental culture with the 5.5 mM glucose concentration, the Ins2^+/+ β-cells were exposed to cycloheximide (Chx; 100 µg/mL); Chx and chloroquine (50–400 µg/mL); Chx and E-64 (25–100 µM); Chx and lactacystin (5–20 µM); or Chx and MG-132 (10–120 µM) for 2 hours with an untreated control. Cellular proteins (30 µg) were separated on16.5% tricine SDS-PAGE under reduced conditions and then examined by C-peptide or β-tubulin antibody on the same membrane. % cont (Pro): percentage of the (average) proinsulin level in individual treatments compared to the untreated control. % cont (Pro/T): percentage of the (average) proinsulin level in individual treatments compared to the untreated control and normalized by β-tubulin. The data were shown as mean. Statistical significances (*P<0.05; **P<0.01) of the normalized proinsulin level between Chx and individual treatments that included Chx were assessed by two-tailed t-test (n = 4).