(A) INS1E cells were incubated for 24 hr ± 20 µM GRP94 inhibitor (PU-WS13). During the last 3 hr, SubAB was added where indicated. Cell lysates were analyzed by immunoblotting for BiP (top panel), and mAb anti-proinsulin (CCI-17) under nonreducing conditions (above red line) or with anti-proinsulin (Proins), anti-insulin (Ins), and anti-cyclophilin B (CypB, loading control) under reducing conditions (below red line). The positions of molecular mass markers are noted. (B) INS832/13 incubated ±active SubAB (1.5 μg/mL, 4 hr) were processed for immunofluorescence with rabbit anti-calnexin (green) or mAb anti-proinsulin (red). (C) Sections of wild-type or LepRdb/db mouse pancreas (C57BL/6) were de-paraffinized and prepared for indirect immunofluorescence. 1) Wild-type islets immunostained with mAb anti-proinsulin (CCI-17, in red), or the Golgi complex labeled with mab anti-GM130 (in green). 2–5) LepRdb/db mice with random blood glucose >500 mg/dL, as follows. 2 and 3) Mice fed ad lib; immunostained for insulin (blue) and mAb anti-proinsulin (CCI-17, red). 4 and 5) Mice fasted overnight, and immunostained as above. The third panel in each case is a merged image of the single-channel fluorescence. (D) Isolated islets from young male LepRdb/db mice (lanes 1, 2, 5 and 6; random blood glucose values shown above) or wild-type C56BL/6 (‘WT’, lanes 4, 8) or a high-fat fed Ins1+/-,Ins2-/- male (lanes 3 and 7, marked with asterisk) were lysed in RIPA buffer and analyzed by nonreducing or reducing SDS-PAGE and immunoblotting with mAb anti-proinsulin (CCI-17, above red line) or guinea pig anti-insulin (below red line). The positions of molecular mass markers are noted. (E) Isolated islets from WT and Nkx2.1-Cre-mediated LepR-KO mice (random blood glucose values shown above) were lysed in RIPA buffer and analyzed under nonreducing or reducing conditions as in panel D. Molecular mass markers are noted. Asterisk denotes lysate from Ins1+/-,Ins2-/- islets as in panel D. The positions of molecular mass markers are noted. Islet lysates from WT and Nkx2.1-Cre-mediated LepR KO mice were also immunoblotted with guinea pig anti-insulin (bottom right) that weakly cross reacts with proinsulin (Proins).