Ripa cell lysis and extraction buffer

Cells were washed with PBS and lysed using RIPA cell lysis and extraction buffer (Thermofisher Scientific; catalog no. 89901) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermofisher Scientific; catalog no. 78440). Total protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermofisher Scientific; catalog no. 23225). Proteins were separated by gradient SDS–PAGE and transferred to PVDF membranes. Blots were blocked in PBS containing 5% milk powder and 0.1% Tween, and then incubated overnight at 4°C with primary antibodies; anti-IFI35 (1:1000, Abcam; catalog no. ab233415) and anti-β-Actin (1:5000, Proteintech; catalog no. HRP-60008). After washing, appropriate HRP-conjugated secondary antibody (SAB; catalog no. L3012) was incubated for 1 hours at room temperature. Chemoluminescence was detected by Pierce ECL Western Blotting Substrate (Thermofisher Scientific; catalog no. 32209) and captured on ChemiDoc™ XRS+ System (Bio-Rad; catalog no. 1708265).

Cultured cells were lysed with RIPA cell lysis and extraction buffer (ThermoFisher) supplemented with Halt^TM Protease and Phosphatase Inhibitor Cocktail (ThermoFisher). Protein concentrations were measured using BCA Protein Assay kit (Solarbio). Protein concentrations were normalized with 5× Native Gel Sample Loading Buffer (NCM biotech) and heated at 100 °C for 10 min. Proteins were separated electrophoretically on 10–15% polyacrylamide gel (Bio-Rad) and transferred to PVDF membrane Trans-Blot Turbo transfer system (Bio-Rad). Before incubating with primary antibodies, the membranes were blocked with PBS containing 5% non-fat milk powder and 0.1% Tween. Then, the membranes were incubated with primary antibodies against IFI35 (1:1000, Abcam, #233415; 1:100, Santa Cruz, #393513), β-Actin (1:5000, CST, #3700S) overnight at 4 °C. After washing with PBST, the membranes were incubated with appropriate HRP-linked secondary antibodies (Anti-Rabbit and Anti-Mouse; Abcam) for 1 h at room temperature. Membranes were washed again and incubated with Pierce ECL Western Blotting Substrate (ThermoFisher) and imaged using ChemiDoc^TM XRS+ System (Bio-Rad).