Mouse anti puromycin

The following antibodies were used for immunofluorescence (IF) and/or immunoblotting (IB) at the indicated dilutions: Rabbit anti-peIF2α (for PLA 1:6000 #44728G Invitrogen, and IB, 1:1000, Cell Signaling), mouse anti-eIF2α (IB, 1:1000, Cell Signaling). peIF4B, p4EBP1, peIF4G (IB:1:2500, cell signaling). Most of the commercially available antibodies that should recognize rodent HRI failed to robustly detect the protein in neurons. Nevertheless, the use of HRI knock-out tissue allowed us to identify in neurons, after immunoprecipitation, an HRI-specific band only with one HRI antibody: 07–728, Millipore. Rabbit anti-actin (1:5000, Abcam), rabbit anti-H3 (1:10000, Abcam) rabbit anti-biotin (IB, 1:1000), mouse anti-puromycin (IB, 1:1000, IF 1:3500, Kerafast,), guinea pig anti-MAP2 (IF 1:1000, Synaptic Systems), rabbit anti- HA-tag (IF 1:2000, Rockland) Goat anti-mouse or anti-rabbit IR680 or IR800 (IB, 1:5.000, Licor), goat anti-guinea pig Dylight405 (IF 1:1000, Dianova), goat anti-guinea pig-Alexa488, and goat anti mouse-Alexa546 or -Alexa488, goat anti-rabbit Alexa647 or -Alexa546 (IF all 1:1000, ThermoFisher).

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.

Detection of newly synthesized proteins by PLA was performed as previously described (5 (link), 10 (link), 45 (link)). Immunostaining using mouse antipuromycin (Kerafast; 1:500) antibody in combination with rabbit anti–β-actin (Abcam; 1:1,000), rabbit anti–PSD-95 (cell signaling technologies; 1:1,000), or rabbit anti-Camk2a (Thermo; 1:1,000) was performed overnight at 4 °C. Following 5× PBS washes, PLA was performed (Sigma). Rabbit PLA^plus and mouse PLA^minus probes were used. PLA was performed according to the manufacture’s guidelines. Following PLA, anti-Map2 immunostaining (guinea pig anti-Map2, Cell Signaling; 1:5,000) was performed to label dendrites. Samples were imaged using a 40× oil objective on a LSM780 or LSM880. Z-stacks (0.43 μm) spanned the entire volume of imaged neurons. Images were analyzed using ImageJ. A 100-μm segment of the dendrite was assessed for the number of Puro-PLA puncta and the density of signal was calculated.