Anti flag ab

For STAT1-FLAG co-ip, cell lysates were incubated with agarose beads conjugated to anti-FLAG Ab (Sigma). Cross-linking chromatin immunoprecipitation (X-ChIP) promoter assays were performed as described previously ^{11 (link)}. Immunoprecipitated DNA was extracted with phenol/chloroform and ethanol precipitation, and a 10% aliquot was analyzed by real time PCR using primers for human IRF1 promoter (5′-CTTCGCCGCTAGCTCTAC-3′ and 5′-CCCATTGGCCGGCTGCGT-3′ as forward and reverse primer pairs and 5′-CAGCCTGATTTCCCCGAAATGACG-3′ as probe). For X-ChIP protein interaction assays, cell lysates were sheared using a 29-gauge syringe instead of sonication and incubated with agarose beads conjugated to anti-p300/CBP Ab (Sigma), washed, and immunoblotted with anti-STAT1, anti-PARP9, anti-DTX3L, or anti-p300/CBP Ab. For co-ip of histone H2BJ with PARP9-DTX3L-STAT1, HEK293T cells were transfected with HA-H2BJ (Life Technologies, CA), FLAG-PARP9 and c-Myc–DTX3L for 48 hours, and cell lysates were immunoprecipitated with anti-HA or c-Myc Ab and immunoblotted with HA-H2BJ, FLAG-PARP, c-Myc-DTX3L, and STAT1 Ab. For X-ChIP assay of DTX3L binding to IFIT1 promoter, cells were infected with EMCV (MOI 1) for 6 hours and then treated with formaldehyde. Cell lysates were incubated with anti-c-Myc Ab, and immunoprecipitated DNA was analyzed by real-time PCR assay using primers for IFIT1 promoter sequence.