Rabbit anti actin

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.

Proteins were separated on Bolt 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to Protran 0.45 NC membranes (GE Healthcare Life Sciences, Chicago, IL, USA) using the Trans-Blot SD semi-dry transfer cell. The membrane was blocked for 1 h in 5% w/v non-fat dry milk in 0.1% Tween-PBS. The membrane was then incubated overnight at 4 °C in 5% w/v non-fat dry milk in Tween-PBS containing either mouse anti-E (Aalto Bio Reagents, Dublin, Republic of Ireland, AZ 1176), rabbit anti-actin (Sigma-Aldrich, St. Louis, MO, USA, A2066), rabbit anti-NS5 [33 (link)], rabbit anti-gamma tubulin (Sigma-Aldrich, St. Louis, MO, USA, T3559), or rabbit anti-GRP78 (Abcam, Cambridge, UK, ab21685). Following 3 × 10 min washes with Tween-PBS, the membranes were then incubated for 1 h in 5% w/v non-fat dry milk in Tween-PBS with anti-mouse or anti-rabbit secondary antibody conjugated with either horseradish peroxidase (HRP) (Abcam, Cambridge, UK) or a near-infrared fluorescent dye (LI-COR Biosciences, Lincoln, NE, USA). This was followed by three 10 min washes with 0.1% Tween-PBS buffer; enhanced chemi-luminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for signal detection of HRP antibodies, whereas fluorescently labelled antibodies were imaged on an Odyssey CLx (LI-COR Biosciences, Lincoln, NE, USA).

Rabbit anti-actin (Sigma), rat anti-IL1-beta/IL-1F2 (R&D), rabbit anti-TGF-beta (Nordic Biosite), rat anti-GM-CSF (Abcam), rat anti-IL10 (Abcam), goat anti-M-CSF (Abcam), goat anti-VEGFD (Abcam), rabbit anti-VEGF (Abcam), rabbit anti-VEGFC (Abcam), rabbit anti-PlGF (Abcam), rabbit anti-IL-6 (Abcam), rabbit anti-phospho (Ser727) STAT3 (Cell Signaling), rabbit anti-phospho, (Tyr705) STAT3 (Cell Signaling), rabbit anti-STAT3 (Cell Signaling). Anti-GM-CSF, IL-10 and IL-1beta were monoclonal antibodies, all other antibodies were polyclonal.

For Western blot analysis cells were lysed in RIPA buffer and different amounts of total proteins were separated by electrophoresis through SDS-PAGE gels and blotted into nitrocellulose membranes. Mouse anti-RTN-1C (Abcam, ab8961) was diluted 1:1000 rabbit anti-LC3 (Cell Signaling, 3868 S) was diluted 1:1000, rabbit anti-syntaxin 17 (Abcam, ab116113) was diluted 1:200, rabbit anti-HA (Sigma, H6908) was diluted 1:2000. Rabbit anti-actin (Sigma, A2066) diluted 1:2000, mouse anti-GADPH diluted 1:1000 (Santa Cruz, sc-47724) and mouse anti-tubulin 1:2000 (Sigma, T-4026) were used as loading controls. Secondary antibodies (Biorad, 1721011, 1706515) were diluted 1:5000.

The AP1 affinity pull-down sample was separated via one-dimensional SDS–PAGE together with the DMSO pull-down sample. After SDS–PAGE, the proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad). The blots were blocked with 5% (w/v) BSA in PBS with 0.1% Tween 20 (PBS-T) for 4 h at room temperature. The membranes were incubated with rabbit anti-actin (1: 500; Sigma), rabbit anti-MSP-1 (1:5,000), mouse anti-PM-I (1:5,000) and rabbit anti-PM-II (1:5,000). Horseradish peroxidase (HRP)-conjugated anti-rabbit (Pierce Biotechnology) or HRP-conjugated anti-mouse IgG (1:5,000; GE Healthcare) was used as a secondary antibody, and samples were incubated for 2 h at room temperature. The membrane was washed three times in PBS-T between each antibody incubation step. Subsequent visualization was performed using ECL substrate (Pierce Biotechnology).

WNV_KUN specific anti-NS1 (clone 4G4; (12)), anti-NS5 (clone 5H1.1) and anti-Envelope monoclonal antibodies were generously provided by Dr Roy Hall (University of Queensland, Brisbane, Australia). WNV_KUN-specific rabbit anti-NS3 polyclonal antisera has been described previously (18). Rabbit anti-Calnexin was purchased from Epitomics. Rabbit anti-Actin and anti-GRP78 were purchased from Sigma. Rabbit anti-PLA2G4A and anti-phosphorylated PLA2G4A were purchased from Cell signaling Technology, and anti-PLA2G4C from Novus Biologicals. Mouse anti-dsRNA (clone J2) antibodies were purchased from English & Scientific Consulting Bt. (Hungry). Alexa Fluor 488- and 594-conjugated anti-rabbit and anti-mouse specific IgG were purchased from Molecular Probes (Invitrogen, Leiden, The Netherlands).