Sodium arsenite (Sigma-Aldrich) was added to the cells at a concentration of 250 μM for 20 min prior to fixation or cell lysate collection. The PKR inhibitor C16 (Sigma-Aldrich) was added to infected cells at a concentration of 1 μM at 1 hpi, and cell lysates were collected at 12 hpi. The ISR inhibitor ISRIB (Sigma-Aldrich) was added at 1 hpi at 0.5 μM. Puromycin (Life Technologies) was added to cells at a concentration of 10 μg/ml at indicated times prior to cell lysate collection. Poly(I⋅C) (Sigma-Aldrich, catalog no. P1530) is a dsRNA analogue and was used as a positive control for immune activation. Poly(I⋅C) was used at a concentration of 20 μg/ml and added to the cell culture medium in combination with Lipofectamine 2000. MNV ORF1 plasmids were described and used previously (22 (link)).
Goat anti-eIF3η, goat anti-G3BP1, and goat anti-TIA-1 were all purchased from Santa Cruz Biotech. Rabbit anti-eIF2α was purchased from Invitrogen; Rabbit anti-actin from Sigma-Aldrich; Mouse anti-Puromycin was obtained from Kerafast, Inc. Mouse anti-G3BP1, mouse anti-GAPDH, rabbit anti-His, and rabbit anti-calnexin were obtained from Abcam, and rabbit anti-p-eIF2α (S52) and Alexa Fluor-conjugated species-specific IgG were purchased from Life Technologies. Rabbit anti-NS7 and rabbit anti-NS5 were manufactured and produced by Invitrogen.
Goat anti-eIF3η, goat anti-G3BP1, and goat anti-TIA-1 were all purchased from Santa Cruz Biotech. Rabbit anti-eIF2α was purchased from Invitrogen; Rabbit anti-actin from Sigma-Aldrich; Mouse anti-Puromycin was obtained from Kerafast, Inc. Mouse anti-G3BP1, mouse anti-GAPDH, rabbit anti-His, and rabbit anti-calnexin were obtained from Abcam, and rabbit anti-p-eIF2α (S52) and Alexa Fluor-conjugated species-specific IgG were purchased from Life Technologies. Rabbit anti-NS7 and rabbit anti-NS5 were manufactured and produced by Invitrogen.