Figure 4

ER stress signaling pathways are activated by insulin 2 (C96Y)-EGFP expression. Clone #4S2 cells were untreated (-Dox) or treated with 2 μg/ml doxycycline (+Dox) for the times indicated. Control INS-1 cells exposed to thapsigargin (Tg) or dithiothreitol (DTT) were used as a positive control. A. RNA was isolated from the cells and XBP-1 cDNA was amplified by RT-PCR. The unspliced form of XBP-1 (uXBP-1, 480 bp) and spliced form of XBP-1 (sXBP-1, 454 bp) are indicated. B. Clone #4S2 cells were treated as in (A), washed in PBS and lysed. Equal amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-phospho-eIF2α and GM130 antibodies. C. Clone #4S2 cells were treated with 1 mM dithiothreitol (DTT) for 30 min. or doxycycline as indicated and fixed for immunofluorescence labeling with anti-ATF6 antibody. Nuclear fluorescence of ATF6 was quantified as described in the methods.