Anti pirf3

Anti-NF-κB, anti-IRF3, anti-TBK1, anti-IFN-β, anti-GAPDH, anti-pNF-κB, anti-pTBk1, anti-pIRF3 and anti-DDDDK for WB and immunofluorescence (IF) assays were purchased from Abcam (Abcam, Cambridge, UK). Anti-HA and anti-Myc for WB and immunofluorescence (IF) assays were purchased from Thermo Fisher (ThermoFisher, Waltham, MA, USA). Anti-J-2 for IF assays were purchased from Scicons (Scicons, Szirák, Hungary). Secondary antibodies labeled with Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 or Alexa Fluor 647 for the IF assay were purchased from Abcam (Abcam, Cambridge, UK). Goat anti-mouse IgG H&L (HRP) and goat anti-rabbit IgG H&L were purchased from Abcam (Abcam, Cambridge, UK) and used in the WB assays.

Total proteins were extracted using cell lysis buffer (BioFeng, Changsha, Hunan, China). After the protein concentrations were determined by BCA kits, the protein samples were extracted and separated by 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skimmed milk and incubated overnight, using the following main detection antibodies at 4°C: anti-GAPDH (1 : 1,000; Abcam), anti-Histon-H3 (1 : 1,000; Abcam), anti-Hsp60 (1 : 1,000; Abcam), anti-cGAS (1 : 1,000; Abcam), anti-STING (1 : 1,000; Abcam), anti-pSTING (1 : 1,000; Abcam), anti-TBK1 (1 : 1,000; Abcam), anti-pTBK1 (1 : 1,000; Abcam), anti-IRF3 (1 : 1,000; Abcam), anti-pIRF3 (1 : 1,000; Abcam), anti-PD-L1 (1 : 1,000; Abcam), anti-CD9 (1 : 1,000; Abcam), anti-TSG101 (1 : 1,000; Abcam), anti-Alix (1 : 1,000; Abcam), anti-Hsp90 (1 : 1,000; Abcam), or anti-β-actin (1 : 5,000; Proteintech). We washed 3 times with TBS-T, and the membranes were cultured with the secondary antibody at 24°C for 1 hr. The western blots were pictured using an ECL Reagent (Pierce, USA), and the density was verified using ImageJ software (NIH, USA).

HEK 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and maintained at 37°C in a humidified 5% CO₂ incubator. Transient transfection reagent Lipofectamine 2000 was purchased from Invitrogen. Anti-HA (MBL), anti-Flag (MBL), anti-V5 (MBL), anti-Myc (MBL), anti-β-Actin (Beyotime), anti-IRF3 (Abcam), and anti-pIRF3 (Abcam) antibodies were used in the study.