Anti pkr

Cells obtained from BAL of WT or Ripk3^-/- mice at day 3 post-infection or BMD-Mφ were lysed in lysis buffer (1% Triton X-100, 150mM NaCl, 20mM Hepes pH7.5, 10% glycerol, 1mM EDTA, supplemented with anti-protease and anti-phosphatase cocktails, Roche) and protein concentration was determined using BCA assay (Pierce). 20 μg of protein were resolved by SDS-PAGE and transferred onto PVDF membranes (Biorad). Membranes were blocked and incubated overnight at 4°C with gentle agitation with primary antibodies. The following primary antibodies were used: anti-RIPK3 (1:1000, Proscience #2283), anti-phospho-PKR (1:200, Santa Cruz Biotechnology #sc-101784), anti-PKR (1:200, Santa Cruz Biotechnology #sc-1702), anti-RIPK1 (1:1000, Cell Signaling Technology #3493), anti-phospho-IRF3 (1:500, CST #4947), anti-IRF3 (1:1000, CST #4302), anti-phospho-eIF2α (1:1000, CST #3597), anti-eIF2α (1:1000, CST #5324), anti-MAVS (1:1000, CST #4983), anti-TBK1 (1:1000, CST #3504), anti-pTBK1 (1:1000, CST #13498), anti-CYPD (1:1000, Calbiochem #AP1035) and anti-actin (1:10000, Sigma-Aldrich #2066). Primary antibodies were followed by HRP-conjugated secondary antibodies and signal was detected using Clarity ECL kit (Biorad) and acquired on Chemidoc MP System (Biorad). Densitometry analyses were performed using ImageJ software (NIH).

Anti-eIF2α, P-eIF2α (Ser51), eIF4G, P-eIF4G (Ser1108), eIF4E, P-eIF4E (Ser209) and Oct4 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Anti-mNanog was obtained from R&D Systems (Minneapolis, MN). Anti-hNANOG and pPKR (Thr446) were obtained from Millipore (Billerica, MA), and anti-Actin (mouse C4) and c-Myc (polyclonal) were obtained from Sigma (St. Louis, MO). Anti-PKR and Nck1/2 were obtained from Santa Cruz Biotechnology (Dallas, TX). Anti-CReP was obtained from ProteinTech (Chicago, IL).

Antibodies and reagents used in this study were as follows: anti-G3BP1 antibody (BD Bioscience, 611126, Franklin Lakes, NJ, USA), anti-PKR (Santa cruz biotechnology, sc-6282, Dallas, TX, USA) anti-RACK1 (Santa Cruz biotechnology sc-17754), anti-phospho-eIF2α (Ser51) (Cell Signaling Technology, 9721S, Danvers, MA, USA), anti-cleaved caspase-3 (Cell Signaling Technology, 9661S), anti-PARP (Cell Signaling Technology, 9542S), anti-Actin (Cell Signaling Technology, 3700S), anti-PKR (Abcam, ab52506, Cambridge, MA, USA), anti-phospho-PKR(Thr451) (Abcam, ab81303), anti-IMP1 antibody (Bethel Laboratories, A303-424A, Montgomery, TX, USA), anti-HA-HRP (Roche, 12013819001, Mannheim, Germany), morusin (root bark of Morus alba) (Biopurity Phytochemicals Ltd., BP0961, Chengdu, Sichuan, China), ISRIB (Millipore sigma, SML0843, Saint Louis, MO, USA), 3-methyladenine (Millipore sigma, M9281), zVAD-FMK (Santa cruz biotechnology, sc-3067).

Cell lysates of Huh-7.5.1 cells transfected with in vitro-transcribed RNA were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by immunoblotting. To detect PKR and its phosphorylated form, rabbit polyclonal anti-PKR (Clone E120; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit monoclonal anti-phospho-PKR, T446 (Clone E120; Epitomics, Burlingame, CA) were used. As a control for the applied proteins, β-actin was also detected with mouse monoclonal anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO). To detect signals, horseradish peroxidase-conjugated anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA) was used.

Doxorubicin was purchased from Sigma-Aldrich (St. Louis, MO). The PKR inhibitor C16 was purchased from Calbiochem (La Jolla, CA). The anti-phospho eIF2α, anti-eIF2α, and anti-CHOP antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti-GAPDH, anti-actin, anti-caspase-3, anti-PKR, anti-Bcl-2, and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The annexin V probe was purchased from Bioacts (Korea). Hoechst 33342 was purchased from Invitrogen (Carlsbad, CA)