Ab194297

RIP experiments were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA). Briefly, cells from all groups were lysed (500 μL per plate) in a modified cell lysis buffer used for western blotting and IP (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, sodium pyrophosphate, β-glycerophosphate, Na₃VO₄, and leupeptin) (Beyotime institute of Biotechnology). After lysis, each sample was centrifuged to clear the insoluble debris and then pre-incubated with 20 μg protein A agarose beads (Beyotime institute of Biotechnology) by rocking for 30 min at 4 °C, followed by centrifugation and transfer to a fresh 1.5 mL tube. The rabbit anti-ac4C antibodies [EPRNCI-184–128] (1:100, Abcam, ab252215) or rabbit anti-NAT10 antibodies [EPR18663] (1:100, Abcam, ab194297) were added and incubated for 90 min before the re-addition of 20 μg of protein A agarose beads to capture the immune complexes. The agarose beads were then washed three times with ice-cold homogenization buffer. The sequences of the RIP-PCR primers are shown in Supplementary Table S1.

Protein samples derived from mouse skins and cells were extracted by RIPA lysis buffer containing phosphatases and proteases inhibitor cocktails (Roche, USA). Protein concentration was determined by BCA protein assay kit (Pierce, USA). For immunoblotting analysis, protein samples were subjected with SDS-polyacrylamide gel electrophoresis and proteins were transferred to PVDF membranes (Millipore). After being blocked, and incubated with primary antibodies and indicated HRP-coupled secondary antibody, membranes were visualized with ECL and images were captured using the Bio-Rad system. Band intensities were detected, normalized, and quantified with the Image Lab 5.0 software (Bio-Rad). The following antibodies were used in this research: NAT10 (Abcam, ab194297, 1:1000 dilution), STAT3 (CST, 30835, 1:1000 dilution), p-STAT3 (Abcam, ab76315, 1:1000 dilution), p-p65 (CST, 3033, 1:1000 dilution), p65 (CST, 8242, 1:1000 dilution), p-FAK(CST, 8556, 1:1000 dilution), FAK (Abcam, ab40794, 1:1000 dilution), Lamin B1 (Abcam, ab133741, 1:1000 dilution), poly-Ubiquitin (CST, 3936, 1:1000 dilution), Fibronectin(CST, 26836, 1:1000 dilution), α-SMA(Proteintech, 14395-1-AP, 1:1000 dilution), α-Tubulin (Beyotime, AT819, 1:5000 dilution), and GAPDH (Beyotime, AG019, 1:5000 dilution).

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)}.