Prl cmv renilla vector

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.

HEK293T and SH-SY5Y cells were transiently transfected for 48 h with pGL3-promotor-SNAP25, pGL3-promotor-VAMP2, pGL3-basic-STX1A, pGL3-control, pGL3-promoter, or pGL3-basic plasmids (150 ng; 96-well plate) (Promega, Fitchburg, WI, USA) using DreamFect Gold (OZ Biosciences, Marseille, Provence-Alpes-Cote d'Azur, France), according to the manufacturer’s protocol. Co-transfection with the pRL-CMV renilla vector (15 ng; 96-well plate) (Promega, Fitchburg, WI, USA) was used as an internal control for transfection efficiency in a 10:1 molar ratio (firefly:renilla). Dual-luciferase assays were performed in 96-well white plates (CELLSTAR® plates—µClear® bottom; Greiner Bio-One, Kremsmünster, Austria) containing 100 µL medium (without 1% antibiotic/antimycotic) with 1.5 × 10⁴ HEK293T cells/mL or 2.5 × 10⁴ SH-SY5Y cells/mL. After 48 h post-transfection, Synergy Mx Microplate Reader (Agilent, Santa Clara, CA, USA) was used to measure the luciferase activity with the Dual-Luciferase Reporter System (Promega, Fitchburg, WI, USA), according to the instructions recommended by the manufacturer.