Rupintrivir

Mouse anti-Flag, mouse anti-HA, mouse anti-β-Actin antibodies and GW5074 were purchased from Sigma-Aldrich (St. Louis, MO). EV71 VP1 polyclonal antibody was prepared by immunizing rabbits with his-tagged VP1 protein. Coelenterazine was obtained from Promega. RIPA lysis buffer and the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) were purchased from Beyotime (Jiangsu, China). Rupintrivir was purchased from Santa Cruz Biotechnology. Lipofectamine® 2000 was purchased from Thermo Fisher Scientific.

Actinomycin D (ActoD) (Sigma-Aldrich) was used at a concentration of 5 µg/mL to induce apoptosis. The pan-caspase inhibitor z.vad.FMK (BD Pharmigen) was used at concentration of 20 µM. Caspase inhibitors were purchased from Calbiochem and used at 4 µM (caspase 8 inhibitor) or as indicated. DMSO only controls were used to account for any cellular or viral alterations induced by the drug vehicle. The HRV 3C protease inhibitor, Rupintrivir (Santa Cruz, SC-208317) was added to infected cells at 1 µM as indicated. Anti-RIPK1 (ThermoScientific, 1860909) or Anti-RIPK1 n-terminal (Cell signalling 3493) antibodies were used to detect full length RIPK1 and its cleavage products. Antibodies to caspase 3 (9662), Poly(A)-binding protein (PABP, 4992) and tubulin (2148) were purchased from Cell Signalling Technologies. Antibodies to poly-histidine tag (11922416001) Green Fluorescent protein (GFP, 11814460001) were purchased from Roche. Viral protein expression was detected with anti-VP2 antibody (QED Biosciences, 18758). Rabbit polyclonal antibody to eIF4G (sc11373) was purchased from Santa Cruz.