Protease inhibitor mixture

Co-IP and Western blot analysis were performed as described previously (46 (link)). HEK293T cells transfected with the indicated plasmids for 24 h were lysed with cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, and 10% glycerol) containing 1 mM PMSF (Beyotime) and 1× protease inhibitor mixture (MedChemExpress). The cell lysates were incubated with anti-FLAG (M2) beads (Sigma; catalog no.: A2220-5ML) overnight at 4 °C on a roller. The immunoprecipitants were subjected to electrophoresis. In addition, to identify the interactions between endogenous proteins, PAMs were noninfected or infected with ASFV (1 MOI) for 36 h. The cell lysates then were incubated with an anti-S273R polyclonal antibody or IgG for 8 h at 4 ˚C, and S273R complexes were captured using protein A + G-Sepharose. For Western blotting analysis, equal amounts of cell lysates and immunoprecipitants were resolved by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (catalog no.: ISEQ00010; Merck-Millipore). After incubation with primary and secondary antibodies as indicated, the membranes were visualized by an Odyssey two-color infrared fluorescence imaging system (LI-COR).

Cells were lysed with 100 μL RIPA lysis buffer (Beyotime, China, #P0013K) containing protease inhibitor mixture (MedChemExpress, America, #HY-K0010) for 30 min at 4 °C followed by ultrasonic-assisted extraction. The cell lysates were then sonicated and centrifuged to obtain the supernatant. 20–40 μg of proteins were separated by SDS-PAGE. Primary antibodies and secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase were then incubated with the blots. Protein bands were visualized on X-ray film (Sigma, America) using an ECL recognition system (Vazyme, China, #SQ201). Antibodies used are shown in Supplementary Table 3.

Cells were lysed with RIPA lysis buffer (Beyotime, China, #P0013K) containing protease inhibitor mixture (MedChemExpress, America, #HY-K0010) for 30 min at 4 °C, followed by ultrasonic-assisted extraction. The cell lysates were sonicated and centrifuged to obtained the supernatant. Part of the supernatants were collected and heated at 100 °C. The heated and unheated proteins were then separated by SDS-PAGE. Coomassie Brilliant Blue dye was used to stain the SDS-PAGE gels to visualize proteins.