Figure 1.

Induction of insulin 2 (C96Y)-EGFP protein expression in pTet-ON INS-1 cells by doxycyline. A-B. pTet-ON INS-1 #46 cells were transiently transfected with pTRE-Tight Ins2 (C96Y)-EGFP construct in the presence or absence of 2 μg/ml doxycycline (Dox) for 48 h. In (A), cells were washed in PBS, lysed and 10 μg of protein were resolved by SDS-PAGE and immunoblotted using antibodies to insulin and GFP. In (B) the cells were washed in PBS, fixed and mounted. GFP fluorescence was visualized using a laser confocal fluorescence microscope. C-D. A stable Ins2 (C96Y)-EGFP expressing INS1 clone (Clone #4S2), generated as described in the methods, was untreated (-Dox) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. The cells were washed in PBS, lysed and an equal amount of protein per condition were resolved by SDS-PAGE and immunoblotted using antibodies to GM130 and GFP (C). In (D), EGFP fluorescence in fixed cells was visualized by confocal fluorescence microscopy.