The human viperin plasmid has previously been described,11 and mutant versions of the plasmid were constructed in pLNCX2, either via mutagenesis PCR utilizing a QuickChange mutagenesis II system

(Stratagene, La Jolla, CA) or via PCR cloning using the HindIII and NotI sites and 5′FLAG tagging the constructs, using the primers listed in Table 1. Transfection PLX4032 clinical trial of all plasmids was performed using Fugene6 (Roche, Nutley, NJ), according to the manufacturer’s recommendations. All experiments involving real-time PCR were performed in 12-well plates with Huh-7 cells seeded at 8 × 104/well, 24 hours before transfection/infection, and performed at least in triplicate. RNA was extracted from cells using Trizol reagent (Invitrogen). First-strand cDNA was synthesized from total RNA, and real-time PCR analysis was utilized to quantitate relative levels of HCV RNA and viperin messenger RNA (mRNA), in comparison to the housekeeping gene, RPLPO. Reaction conditions click here and primers are expressed as described previously.11 Huh-7 cells were seeded on 0.2% gelatin-coated coverslips in 24-well trays (4 × 104 cells/well) 24 hours before transfection/infection. Cells were either fixed using methanol/acetone

(1:1) for 5 minutes on ice for standard microscopy or with 4% paraformaldehyde for 10 minutes on ice, followed

by a 10-minute incubation in 0.1% Triton-X in phosphate-buffered saline (PBS) for confocal Farnesyltransferase microscopy, before incubation with primary antibodies for 1 hour at room temperature (RT). Cells were washed in PBS and incubated with secondary antibodies for 1 hour at RT before being mounted with Prolong Gold reagent (Invitrogen). Images were acquired with a Bio-Rad Radiance 2100 Confocal (Bio-Rad, Hercules, CA) or a Nikon TiE inverted microscope (Nikon, Tokyo, Japan). Acceptor photobleaching was carried out essentially as previously described19 with the use of Alexa 555– (Invitrogen) and Cy5 (Jackson Laboratories, Westgrove, PA)-conjugated secondary antibodies or GFP- and mCherry-tagged protein constructs. Images of the acceptor and donor flurophores were acquired using a Zeiss Axioplan2 upright microscope, using a 63× PlanApo objective (Carl Zeiss AG, Oberkochen, Germany). Acceptor photobleaching was performed at maximum light intensity for 30-180 seconds, followed by reimaging of the donor and acceptor fluorophores (this was an automated process ensuring identical imaging conditions). The fluorescence energy resonance transfer (FRET) signal (increase in signal postbleach) was determined by the subtraction of the pre- from postbleach donor image using ImageJ software.