Mouse anti actin clone ac 74

Whole cell lysates were prepared using a modified RIPA buffer (20 mmol/L Tris-HCl (pH 7.5) 350 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na3VO4, 1 mg/mL leupeptin). Samples were supplemented with complete protease inhibitor (Roche) and were sonicated followed by centrifugation. Supernatants were recovered and used for Western blotting according to standard procedures. Primary antibodies: mouse anti-actin (clone AC-74, Sigma, A2228), mouse anti-γH2AX (ser139) (JBW301, Millipore, 06-536), rabbit anti-p-p53 (ser15) (Cell Signalling, 9284), rabbit anti-Cleaved caspase-3 (Asp 175) (Cell Signalling, 9661), mouse anti-PARP-1 (Calbiochem, AM30), rabbit anti-PARP (Cell Signaling, 9542), rabbit anti-p21 (Cell Signaling, 2947), rabbit anti-p21 (C-19, Santa Cruz, 397), mouse anti-α-tubulin (Calbiochem, DM1A), rabbit anti-Caspase-3 (Cell Signaling, 9662). After extensive washing primary antibodies were incubated with the specific secondary antibodies bound to the HRP enzyme (horseradish peroxidase-conjugated antibody) (GE Healthcare) and subjected to chemiluminescence using the ECL system (GE Healthcare).

The antibodies used for immunostaining were mouse anti-E glycoprotein (4G2) kindly provided by P. Despres (PIMIT, Université de La Réunion-INSERM France) and anti-Mouse IgG (H + L) Alexa Fluor 555 Secondary Antibody (Life Technologies). The antibodies used for western blot detection were anti-ISG15 (Santa Cruz), anti-flag (clone M2; Sigma-Aldrich) and mouse anti-actin (clone AC74; Sigma-Aldrich). The antibodies used for FACS analysis were APC-conjugated anti-CD163 (clone REA812, MACS Miltenyi Biotec), PE-conjugated mouse anti-HLA-DR (clone LN3, Invitrogen); PE-Cy7-conjugated mouse anti-CD11b (clone ICRF44, BD Biosciences), Pacific Blue-conjugated mouse anti-CD14 (clone 63D3, Biolegend). Other reagents include TLR3 agonist poly(I:C) (Invivogen); Hoescht (Life Technologies) and cDNA synthesis and qPCR kits (Life Technologies). The reagents for the preparation of the libraries include: Vivaspin unit 100 MWA (Sartorius); Pico Total RNA kit (Agilent), T4 RNA ligase (New England Biolabs); Superscript III (Life Technology); Ultra DNA Library Prep kit for Illumina (New England Biolabs): AMPure XP beads (Agencourt); and High Sensitivity DNA kit (Agilent).