Effect of IRE1 inhibitor 4μ8c on mutant proinsulin expression, XBP1 splicing and eIF2α phosphorylation. A. Insulin 2 C96Y-GFP cells were treated or not with 2 μg/ml doxycycline (Dox) for 24 h in the presence or absence of the indicated concentrations of the IRE1 inhibitor 4μ8c. Cell lysates were prepared and immunoblotted for GFP to detect the mutant proinsulin, cleaved caspase 3 and loading control protein GM130. B. Insulin 2 C96Y-GFP cells were treated or not with 2 μg/ml Dox for 48 h in the presence or absence of 5 μM 4μ8c and total RNA was isolated. In lanes 5 and 6 the cells were incubated in 1 μM thapsigargin (Tg) for 1 h prior to RNA isolation. Unspliced (u) and spliced (s) XBP1 cDNA were amplified by RT-PCR. Result is representative of 3 independent experiments. C. Insulin 2 C96Y-GFP cells were treated as indicated and immunoblotted with phospho-eIF2α and GM130 antibodies.
Zhang et al. BMC Cell Biology 2014 15:29 doi:10.1186/1471-2121-15-29