Samd9

Cell lysates (20 μl) were resolved by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Sambrook, 2001 ; Sambrook and Russell, 2006 (link)), transferred to nitrocellulose membranes and blocked with Tris-buffered saline and 0.1% Tween-20 (TBS-T) supplemented with 5% non-fat dried milk for 1 h at room temperature. Subsequently, membranes were incubated with the monoclonal or polyclonal antibody against the following proteins SAMD9 (Sigma), SAMD9L (Abcam), tubulin (abcam), MxA (Abcam), PKR (SANTA CRUZ^®), IFIM3 (Abcam), tubulin (Abcam) overnight at 4°C. Next day, the membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham) and analyzed with chemiluminescence reagents according to the manufacturer’s instructions.

Lysis buffer for standard western blot contains 1% NP-40, 50 mM Tris-Cl pH7.4, 150 mM sodium chloride with protease inhibitor (Millipore Sigma, cOmplete ULTRA Tablets EDTA-free Protease inhibitor, Catalog # 6538282001) as described before [26 (link)]. For detection of phosphorylated proteins, phosphatase inhibitors are added to the base lysis buffer including 2 mM sodium orthovanadate (Santa Cruz, Catalog # 13721-39-6), 1 mM Phenylmethylsulfonyl fluoride (PMSF) (Millipore-Sigma, Catalog # 52332), and 25 mM β-Glycerophosphate (Sigma-Aldrich, catalog# G9422) as described previously [27 (link)]. Antibodies used in this study are β-actin (Sigma-Aldrich, A1978), SAMD9 (Sigma-Aldrich, HPA021319), cGAS (Cell Signaling Technology, 15102S), Phospho-IRF3 (Cell Signaling Technology, 37829S), IRF3 (Cell Signaling Technology, 11904S), FLAG (Sigma-Aldrich, F1804-1MG), RIG-I (Cell Signaling Technology, 3743), MDA5 (Cell Signaling Technology, 5321), V5 (Life Technologies, R960-25). Antibodies for MYXV M040, M038, M062, and M063 are custom made by BIOMATIK Corporation (Delaware, USA).