Puno irf3sa

iSLK.219 (doxycycline-inducible SLK cells harboring latent rKSHV.219) (a kind gift from D. Ganem) were maintained in DMEM (Corning) supplemented with 10% FBS (Sigma), 1% penicillin and streptomycin (Corning), G418 (250 μg/ml) (Sigma), hygromycin (400 μg/ml) (Corning), and puromycin (10 μg/ml) (Corning). BCBL-1 cells (a kind gift from D. Ganem) were maintained in RPMI (Corning) medium supplemented with 20% FBS, 1% penicillin and streptomycin (Corning), 1% L-glutamine (Corning), and 0.05 mM β-mercaptoethanol (Sigma). All cells were maintained at 37°C in a 5% CO2 laboratory incubator subject to routine cleaning and decontamination. poly(I:C) was purchased from Invivogen. Antibodies were obtained from the following sources: mouse anti-NLRX1 (Jenny Ting laboratory), KSHV ORF45 (MA5-14769) (Thermo Scientific), Goat anti β-actin-HRP (1615) (Santa Cruz), KSHV K8.1alpha (SC-57889; Santa Cruz). The NLRX1 antibody was previously reported [12 (link)]. The pCIG2-Puro and pCIG2-NLRX1-FLAG plasmids were previously described [17 (link)]. The dRIG-I and MAVS expression plasmids were previously described [12 (link)]. pRL-CMV renilla vector was obtained from Promega. IFNβ promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen.

pcDNA4/TO-ORF48-Strep was a kind gift from Britt A. Glaunsinger at the University of California, Berkeley. HA-PPP6C was a kind gift from David L. Brautigan at the University of Virginia. PPP6C-FLAG and HA-PPP6C truncated mutants were cloned into pcDNA3.1 (Invitrogen) using a standard molecular cloning protocol. PPP6C H55Q, H114Y, and G189R mutants were generated using a QuikChange Lightning site-directed mutagenesis kit (Agilent). HA-STING, HA-STING-N (amino acids [aa] 1 to 195) and HA-STING-C (aa 181 to 379) were kind gifts from Glen Barber at the University of Miami. pRL-TK plasmid was obtained from Promega. IFN-β-luc plasmid was a generous gift from Zhijian Chen at the University of Texas Southwestern Medical Center. pCIG2-PURO-FLAG, ΔRIG-I, MAVS, and TBK1 plasmids were kind gifts from Jenny Ting at the University of North Carolina at Chapel Hill. pUNO1-cGAS and pUNO-IRF3sa were purchased from InvivoGen.