DNase
treatment was performed on the eluted RNA to avoid residual DNA contamination. Eight hundred nanograms of the eluted RNA was subjected to reverse transcription by a commercial kit (Superscript™ III; Invitrogen, Carlsbad, CA, USA), following the manufacture’s instructions, and then subjected to PCR amplification using the primers pairs for UL54 and UL13 as described Kleymann et al. (2002) and Tal-Singer et al. (1997) and for UL52 and ACTB (β-actin) as described by Su et al. (2008). The PCR reaction was carried out in a final volume of 25 μl containing 20 mM Tris–HCl (pH 8.5), 50 mM KCl, 1.5 mM Afatinib concentration MgCl2, 0.2 mM of each deoxynucleoside triphosphate, 0.2 μM of each specific primer, 2.5 U of GoTaq DNA polymerase (Promega, Madison, WI, USA), and genomic DNA or cDNA. The PCR program for UL52, UL13 and UL54 and ACTB consists of denaturation at 94 °C for 5 min and 30 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 40 s, and polymerization at 72 °C for 40 s,
followed by a final extension at 72 °C for 10 min. The expected sizes for UL54, UL13, UL52 and ACTB are 283, 600, 259 and 314 bp, respectively. Five-microliter aliquots of the PCR products were resolved on a 1.5% agarose gel. Vero cell monolayers were infected with HSV-1 at MOI 0.2 for 1 h. Next, residual viruses were removed with PBS and cells received different treatments for 18 h. Then, cells were trypsinized and MDV3100 lysed with lysis buffer [0.125 M Tris–HCl (pH 7.4), 30% glycerol, 100 μg/ml phenylmethylsulfonyl fluoride, 2% sodium dodecyl sulfate and 5% β-mercaptoethanol]. Cell lysates were clarified by centrifugation, and proteins were denatured by boiling, and equivalent amounts of protein (5 μg) were separated on 12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk in blotting buffer [25 mM Tris–HCl
(pH 7.4), 150 mM NaCl, 0.1% Tween 20]. All membrane washing steps were performed using this blotting buffer. The membranes were incubated for 90 min with the following primary antibodies: CYTH4 goat monoclonal antibody against ICP27 protein (1:700 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse monoclonal antibody against UL42 protein (1:5000 dilution) (Millipore); mouse monoclonal antibody against gD (1:5000 dilution) (Santa Cruz Biotechnology), mouse monoclonal antibody against gB (1:5000 dilution) (Millipore); rabbit monoclonal antibody against β-actin (1:5000 dilution) (Millipore). After washing, the membranes were incubated with the respective secondary antibodies for 1 h. The immunoblots were developed and detected using the Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA), according to the manufacture’s instructions.