Figure 1

Down-regulation of P-eIF2α and P-PKR by PC1. (A) Schematic presentation of human PC1 membrane topology, showing 11 transmembrane domains, extracellular N- and intracellular C-termini. The numbers and positions of the starting amino acids for three truncation mutants are indicated. (B) Representative WB data. 60 µg of total proteins from HEK293T cells transfected with PC1-5TMC or GFP vector were loaded for immunoblotting. Blots were probed with the indicated antibodies. β-actin served as loading controls. Control (Ctrl), GFP vector; 5TMC, GFP-tagged PC1-5TMC. (C) Statistical data showing the relative activities (%) of eIF2α and PKR in HEK293T cells from panel B assessed by P-eIF2α/eIF2α and P-PKR/PKR, respectively. Shown are averaged P-eIF2α/eIF2α (N = 15, P = 0.002, paired t-test) and P-PKR/PKR (N = 10, P = 0.001, paired t-test). (D) Representative WB data using HeLa cells from similar experiments as in panel B. (E) Representative WB data using native HEK293T cells and those stably expressing mouse WT PC1. Cell were collected and loaded for immunoblotting by the indicated antibodies. Ctrl, native HEK293T cells; PC1, HEK293T cells stably expressing Flag-tagged full-length PC1. (F) Statistical data showing averaged ratios (%) of P-eIF2α/eIF2α (N = 9, P = 0.007, paired t-test) and P-PKR/PKR (N = 10, P = 0.002, paired t-test) from panel E. (G) Representative WB data showing the expression of P-eIF2α and P-PKR in mouse Pkd1 knockout mouse embryonic kidney epithelial cells. +/+, WT; −/−, Pkd1 homozygote. (H) Averaged and normalized ratios (%) of P-eIF2α/eIF2 and P-PKR/PKR from panel G are plotted. *p = 0.04, **p = 0.002 (N = 4, paired t-test).