Anti eif2α antibody

The embryos were deyolked and the proteins were extracted by lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 5 mM EDTA, 10% glycerol and 0.1% Triton X-100) containing appropriate protease inhibitors (Bimake; B14001) and phosphatase inhibitor cocktail (Bimake; B15001). The boiled protein samples were first analyzed by SDS-PAGE and Coomassie blue staining as a preliminary normalization^{100 (link)}. Immunoblot analysis was performed with antibodies including anti-phospho-eIF2α (Ser51) (D9G8) XP rabbit monoclonal antibody (mAb) (Cell Signaling Technology; 3398), anti-eIF2α antibody (Cell Signaling Technology; 9722), anti-phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb (Cell Signaling Technology; 9234), anti-p70 S6 Kinase (49D7) Rabbit mAb (Cell Signaling Technology; 2708) and anti-β-actin mouse mAb (YEASEN; 30101ES60).

Mouse tissue or cultured NRCMs were homogenized in lysis buffer (cell signaling technology, #9803) with 1 mM phenylmethylsulfonyl fluoride (PMSF), and lysates were separated on 5–20% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membranes were probed with one of the following primary antibodies overnight at 4 °C: anti PERK antibody (1:200, Santa cruz), anti p-PERK (Thr980) antibody (1:100, cell signaling technology), anti eIF2α antibody (1:1000, cell signaling technology), anti p-eIF2α (Ser51) antibody (1:1000, cell signaling technology), anti PRL (N-terminal) antibody (1:1000, Acris Antibodies GmbH), anti phosphorylated STAT3 antibody (1:2000, cell signaling technology), anti STAT3 antibody (1:1000, cell signaling technology), anti cleaved caspase3 antibody (1:1000, cell signaling technology), anti GAPDH antibody (1:1000, cell signaling technology), and anti transferrin antibody (1:1000, Santa cruz).
This was followed by a 1-h incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) at room temperature. Bound antibodies were detected by chemiluminescence with the ECL detection system. Relative protein levels were quantified using the Image J program (NIH, Bethesda, MD). Some of images (Figs. 1A,F, 3D) did not include full length membranes, with membrane edges visible.

PAMs were infected with ASFV (MOI = 5) for 2 h at 37 °C and washed three times with PBS before incubation with fresh medium. Cells were heat-shocked for 20 min at 50 °C or not treated before collected at 24 hpi for detection by Western blot with anti-eIF2α antibody (1:2000, Cell Signaling Technology, Danvers, MA, USA) and anti-Phospho-eIF2α (Ser51) antibody (1:2000, Cell Signaling Technology) as described above.
SiRNAs used to knock down G3BP1 (si-G3BP1: CAUUAACAGUGGUGGGAAA) and G3BP2 (si-G3BP2: GGAGCAAGAAGAAAGACAA) were designed and synthesized by Biotend, Shanghai. 50 nM si-G3BP1 and 50 nM si-G3BP2 or 100 nM negative control siRNA (si-nc) were transfected into 3D4/21 cells using lipfectamine 3000 (Invitrogen) according to the instructions for 12 h before subsequent ASFV infection at 5 MOI. Cells were collected at 48 hpi for detection by western blot with anti-G3BP1 antibody (1:2000, GeneTex, Irvine, CA, USA) and anti-G3BP2 antibody (1:2000, Abcam, Cambridge, UK) as described above.