Figure 1

A model of PKR with its protein-binding function but lacking kinase catalytic activity was built in pancreatic β cells. MIN6 cells were transfected with plasmids encoding GyrB-PKR-K296H or the empty vector as indicated. Twenty-four hours after transfection, cells were treated with coumermycin for 24 h and cell extracts were collected. (A) The first three lines are mock, 2-AP and BEPP, and others were treated as indicated. Cell extracts were analyzed by Western blotting using antibodies against PKR, p-eIF2α and eIF2α. (B) qRT-PCR performed to detect the mRNA levels of TRAF2, −3, −5 and -6 in MIN6 cells. (C) Extracts prepared as described for panel A (the last four lines) were immunoprecipitated with antibodies against PKR. Immunocomplexes or whole cell extracts (WCE) were analyzed by Western blotting with antibodies against TRAF2, −3 and −6. Asterisks denote IgGs, and arrows indicate the specific proteins. (D) Isolated mouse islets were administered as MIN6 cells. IFA was performed with antibodies directed against PKR (red) and insulin (green); DAPI was used for nuclear staining (blue). Insulin with green staining was used to identify β cells (scale bar = 100 µm). β-actin was detected as an internal control. Data are means ± SEM of three separate experiments. *P< 0.05 versus control.