Fig. 1
ATF6 cleavage was common among different picornaviruses. a RD cells were mock infected (M) or infected (V) with different strains of EV-A71 and echovirus 9 at an MOI of 10. As outlined in the upper panel, cells were infected with virus for 1 h at - 1 h p.i., after which the unbound virus was removed and replaced with DMEM containing 2% FBS at 0 h p.i. Cell lysates were harvested at 2, 4, 6, and 8 h p.i. and were evaluated by western blotting using an antibody specific for endogenous ATF6. Anti-GAPDH was used as an internal loading control. b Assessment of p90ATF6 level in EV71-infected cells by pulse–chase and immunoblotting assays. RD cells were infected with EV71 (strain 2231) or treated with (10 μg/ml) cycloheximide (CHX), and bulk proteins were metabolically labeled with [35S]methionine/cysteine for 1 h before harvest at the time points indicated. Cell lysates were collected and analyzed by SDS-10%PAGE and autoradiography (panel c). The basal level of p90ATF6 was detected by western blotting using antibodies against ATF6 (panel a). GAPDH was used to monitor equal loading (panel b). c EV71-infected p90ATF6-overexpressing RD cells were harvested at 7 h or were treated with 2.5 mM DTT for 7 h and evaluated by western blotting using an antibody specific for the HA tag. DTT-treated cells were used as a positive control for ATF6 cleavage under conditions of definitive UPR induction. The products of ATF6 cleavage induced by viral infection and DTT are indicated by an arrowhead and an asterisk, respectively