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. 2012 Dec 7;288(3):1896–1906. doi: 10.1074/jbc.M112.420018

FIGURE 3.

FIGURE 3.

Trafficking of hPro-CpepSfGFP in GRINCH cells. A, GRINCH cells were incubated with cycloheximide (10 μg/ml, for protein synthesis inhibition) for the indicated times. At each time the cells were lysed, and the samples were resolved by reducing SDS-PAGE in 4–12% acrylamide gradient gels with electrotransfer to nitrocellulose for Western blotting (WB) with anti-GFP (upper panel), anti-insulin (center panel), or anti-tubulin (lower panel). Note the disappearance of both hPro-CpepSfGFP and proinsulin over the 4-h time course. B–D, images obtained by confocal microscopy. Scale bar = 20 μm. B, GRINCH cells were plated on chamber slides and were incubated 48 h later without (Con) or with cycloheximide (+CHX) for 4 h before fixation. The cells were processed for immunofluorescence with anti-insulin (blue) and anti-calnexin (red). C, the same cells were processed with anti-proinsulin (red). D, INS1 cells were transiently transfected with hProC(A7)Y-CpepSfGFP, fixed, and processed with anti-proinsulin. Endogenous proinsulin is clearly juxtanuclear in untransfected cells and shifts to an ER staining pattern in cells coexpressing misfolded mutant proinsulin.