Anti stat1 sc 346

Anti-MuV V/P (T61) and L (L17) and anti-MeV P rabbit polyclonal antibodies (PAbs) were described previously [37 (link),49 (link),52 (link)]. Anti-MuV N rabbit PAb was provided from DENKA SEIKEN (Niigata, Japan). Anti-FLAG (M2) and anti-β-actin (AC-15) mouse monoclonal antibodies (MAbs) were purchased from Sigma (St. Louis, MO). Anti-STAT1 (sc-346) and anti-STAT2 (sc-476) rabbit PAbs were purchased from Santa Cruz Biotechnology (Dallas, TX). Anti-HA (HA11), anti-PIH1D1 (ab57512), anti-Hsp90 (Hyb-K41009) and anti-IRF3 (SL-12.1) mouse MAbs and anti-MeV N (NB100-1856), anti-RuvBL1 (#12300) and anti-RPAP3 (A304-854A) rabbit PAbs were purchased from BioLegend Inc. (San Diego, CA), Abcam (Cambridge, UK), StressMarq Bioscience Inc. (Victoria, Canada), BD Pharmingen/BD Bioscience (San Jose, CA), Novus Biologicals (Littleton, CO), Cell Signaling Technology (Danvers, MA) and Bethyl Laboratories Inc. (Montgomery, TX), respectively.

Plasmids encoding ZIKV NS1, NS2B3 and NS4B were chemically synthesized based on Z1106033 ZIKV strain (NCBI ID: KU312312) and sub-cloned into pcDNA3.1 expression vectors using standard molecular biology techniques. RIG-I (2CARD), MDA5, MAVS, TBK1, IRF3 (5D) and IFNβ, ISRE, NF-κB luciferase reporter plasmids have been previously described [42 (link)]. Other plasmids mentioned were acquired by the means of standard PCR techniques. Horseradish peroxidase anti-Flag (M2; A8592) and anti-β-actin (A1978) were purchased from Sigma. Horseradish peroxidase anti-hemagglutinin (HA; 12013819001) was purchased from Roche Applied Science (Shanghai, China). Anti-pTBK1 (5483), anti-TBK1 (3013), anti-pIRF3 (4947s), anti-Jak1 (3344), anti-pJak1 (3331), anti-pSTAT1(9167), anti-C-PARP (5625), anti-C-Caspase 3 (9661), anti-p62 (8025) and anti-Beclin-1 (3738) were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-IRF3 (sc-9082) and anti-STAT1 (sc-346) were from Santa Cruz Biotechnology (Dallas, TX, USA).

ChIP experiments were performed exactly as previously described8 (link). Briefly, nuclear extracts from 5 × 10⁶ or 10⁷ neutrophils (for ChIP targeting, respectively, H3K27Ac or STAT1, IRF1, C/EBPβ and PolII) were immunoprecipitated with 1 μl anti-H3K27Ac (ab4729) (Abcam, Cambridge, United Kingdom), 25 μl anti-STAT1 (sc-346), 25 μl anti-IRF1 (sc-497), 20 μl anti- C/EBPβ (sc-150), 20 μl anti-PolII (sc-899) (Santa Cruz Biotechnology). To establish the background levels of ChIP experiments, the precipitation signal was quantified also at the promoter of prolactin (PRL) since it is completely silent in myeloid cells. The coimmunoprecipitated material was subjected to qPCR analysis using the following promoter specific primers (purchased from Life Technologies): IL-6 forward: 5′-TAGCCTCAATGACGACCTAAG-3′; IL-6 reverse: 5′-GTGGGGCTGATTGGAAACCT-3′; IFIT1 forward: 5′-GGCAGCAATGGACTGATGTTC-3′; IFIT1 reverse: 5′-GGAAACCGAAAGGGGAAAGTG-3′; and PRL forward: 5′-AGGGAAACGAATGCCTGATT-3′; PRL reverse: 5′-GCAGGAAACACACTTCACCA-3′. Data from qPCR were expressed as percentage over input DNA and are displayed as means ± SEM.