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.
Hartley et al. BMC Cell Biology 2010 11:59 doi:10.1186/1471-2121-11-59