Anti peif2αs51

Western blotting has previously been described (18 (link)). Anti-pEIF2α^S51, anti-EIF2α, anti-ATF4, anti-GRP78, anti-IRE1α, anti-acetylated-α-tubulin, anti-BCL2, anti-BCLXL, anti-PUMA, anti-BID, anti-BIM (Cell Signaling Technology, Beverly, MA, USA) and anti-pIRE1α^S724 (Abcam, Cambridge) were used in conjunction with a HRP-conjugated anti-rabbit secondary antibody (Amersham, Buckinghamshire, UK). Anti-caspase-8 (12F5; Alexis, San Diego, CA, USA), anti-CHOP (Cell Signaling Technology), anti-ATF6 (Abcam), anti-MCL1 (BD pharmingen, Oxford, UK) and anti-NOXA (Abcam) mouse monoclonal antibodies were used in conjunction with a horseradish peroxidase–conjugated anti-mouse secondary antibody (Amersham).

Cells were collected as previously described using a mixture of T-PER reagent (Pierce, IL), 2× Tris-glycine (sodium dodecyl sulfate) SDS sample buffer (Novex, Invitrogen), 33 mM dithiothreitol (DTT), and protease/phosphatase inhibitor cocktail tablets (1× Halt cocktail, Pierce) applied directly to cells prewashed in PBS ^{27 (link)}. Samples were then scraped, collected and boiled for 10 minutes. Cell lysates were separated in 4–12% Bis-Tris gels and transferred overnight using a wet transfer on PVDF. The membranes were blocked with either a 3% BSA or 3% boiled milk solution in PBS with Tween20 (PBS-T) for an hour at room temperature. Membranes were probed with anti-RVFV N, anti-RVFV Gn (#4519, ProSci), anti-V5 (Serotec), anti-Flag (Sigma Aldritch), anti-PP1α and β (Cell Signaling), anti-PP1β (Abcam), anti-peIF2α (S51) (Cell Signaling) and HRP conjugated actin, diluted in blocking buffer, then incubated overnight at 4°C. The blots were then washed four times with PBS-T and incubated with either secondary HRP-coupled anti-goat, anti-rabbit or an anti-mouse antibody diluted in blocking buffer. The blots were visualized by chemiluminescence using the SuperSignal West Femto Maximum Sensitivity substrate kit (Thermo Scientific) through the Molecular Imager ChemiDoc XRS system (Bio-Rad).