Figure 7

RIP1, located downstream of TRAF2, is implicated in PKR protein-binding-mediated NF-κB activation and β-cell proliferation in MIN6 cells. (A) Administered as Figure 1B, MIN6 cell extracts were collected for immunoprecipitation with antibodies against RIP1 and thoroughly washed. Immunocomplexes were analyzed by Western blotting with antibodies against ubiquitin and whole cell extracts (WCE) with anti-RIP antibody. (B) si-NC or si-TRAF2 was transfected into MIN6 cells before GyrB-PKR-K296H plasmids. Immunoprecipitation and Western blotting were performed as in panel A. (C) Twenty-four hours after transfection of si-RIP1, luciferase reporter assay was conducted as in Figure 6A, and fold luciferase activity is shown (left panel); Western blot to detect c-myc protein level is described (right panel). (D) Quantification of EdU-positive β cell was shown. β-actin was detected as an internal control. Data are means ± SEM of three separate experiments. *P < 0.05 versus control.