Western Blot Analysis of Protein Expression

Cells were lysed with RIPA lysis buffer (1 × SDS, 1 × phosphatase inhibitor cocktail, 1 × protease inhibitors) to obtain protein samples. Then, the samples were separated with SDS-PAGE and transferred to the PVDF membrane. The blots were visualized by ECL after incubating with the primary antibodies and secondary antibodies. Anti-GAPDH (sc-47724; 1:500), anti-β-actin (sc-47778; 1:500) and anti-Tubulin (sc-23948; 1:500) were purchased from Santa Cruz Biotechnology, while anti-Flag was purchased from MBL (M185-3L, Tokyo, Japan; 1:1000). Antibodies against NAT10 (ab194297; 1:1000), LANA (ab4103; 1:1000), IFI16 (ab169788; 1:1000) and ac⁴C (ab252215; 1:1000) were from Abcam. Anti-pro Caspase-1+p10+p12 (ab179515; 1:1000) was also obtained from Abcam. IL-1β (3A6) mouse mAb (12242; 1:1000) and cleaved Caspase-1 (Asp296) (E2G2I) rabbit mAb (89332; 1:1000) were from Cell Signaling Technology. The monoclonal rabbit anti-vIL-6 antibody (1:500) was kindly provided by Dr. Robert Yarchoan from the Center for Cancer Research, National Cancer Institute (Bethesda, Maryland, USA)^{53 (link),54 (link)}, and polyclonal rabbit anti-vIRF1 antibody (1:300) was from Dr. Gary Hayward from Viral Oncology Program, The Johns Hopkins School of Medicine (Baltimore, Maryland, USA)^{55 (link)–58 (link)}.