Perk inhibitor gsk2606414

PERK (#5683), eIF2α (#5324), p-eIF2α (#9721), CHOP (#2895), IRE1α (#3294), XBP1s (#27901), GRP78 (#3177), cleaved PARP (#9532), Bax (#2772), Bcl-2 (#2872) and β-actin (#9562) primary antibodies were purchased from Cell Signaling Technology (CST, MA, US). p-PERK (DF7576) antibody was purchased from Affinity Biosciences (Cincinnati, OH, USA). ATF6 (sc-166659) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, US). ATF4 (ab184909) and p-IRE1α (ab243665) were purchased from Abcam (Burlingame, CA, US). UBA1 inhibitor TAK-243 was obtained from CSNpharm (CSNpharm, Chicago, IL, US). GRP78 inhibitor HA15 and PERK inhibitor GSK2606414 were purchased from MedChemExpress (Shanghai, China). TAK-243, HA15 and GSK2606414 were dissolved in DMSO to create a 10 mmol/L solution, which was diluted to different concentrations in DMEM medium before use.

Vero E6 cells were infected with PEDV at multiplicity of infection (MOI) of 0.001. After adsorption at 37 °C for 2 h, the supernatant was removed. The cell monolayers were washed with sterile phosphate buffered saline (PBS) at pH 7.4 to remove unbound viruses and then incubated in DMEM supplemented with 0.2% trypsin (0.25%; Gibco) at 37 °C for indicated time points.
Vero E6 cells were infected as described above and cultured in fresh medium in the absence or presence of the following chemical: 5 μM PERK inhibitor GSK2606414 (MedChemExpress, Jersey, NJ, USA), 5 μM ERO1α inhibitor EN460 (MedChemExpress), 10 mM antioxidant N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, St. Louis, MO, USA), or the solvent control (dimethylsulfoxide, DMSO). The cells were harvested at 36 h post-infection (hpi) for Western blotting, and the supernatants were harvested for viral titration.