Figure 4

Differential effects of SUMO1 and SUMO3 on PKR and eIF-2α phosphorylation. HeLa-wt, HeLa-SUMO1 and HeLa-SUMO3 cells were transfected with poly(I:C) (a), infected with VSV (b) or EMCV (c) at an MOI of 0.2 for 8 h. Transfected and infected cells were analyzed by Western blot for pPKR and peIF-2α expression. Equal amounts of cell extracts were analyzed with anti-PKR, anti-eIF-2α and anti-Actin antibodies to quantify protein levels. (d) HeLa-wt and HeLa-SUMO1 cells were not transfected, transfected with siRNA scramble or siRNA targeting Ubc9. Cell extracts were analyzed by Western blot for Ubc9, SUMO1 (left panel), PKR, pPKR, peIF-2α, eIF-2α and Actin (right panel). (e) HeLa-wt cells were not transfected, transfected with 2 μg of empty vector pcDNA3 (EV) or with 0.5 or 2 μg of SUMO1. Cell extracts were analyzed by Western blot for PKR, pPKR, peIF-2α, eIF-2α and Actin. Uncropped images of blots are shown in Supplementary Figure 3.