Western Blot Analysis of HCMV Proteins

Western blot was performed on triplicate infections carried out in confluent HFF in 6-well plates at the indicated MOI and treatment conditions. Sonicated whole-cell extracts in lysis buffer containing phosphatase and protease inhibitors were prepared as previously described (44 (link)). The gel-loading buffer contained 2% SDS and 100 mM beta-mercaptoethanol. The proteins were fractionated on freshly made 8% SDS-PAGE Tris-glycine gels prior to transfer to Amersham Protran 0.45-μm nitrocellulose membranes (GE Healthcare Life Sciences 10600002). HCMV IE1-72 and IE2-86 were detected by murine monoclonal antibody MAB810 (EMD Millipore, 1:1,000 dilution). Host actin was detected with polyclonal rabbit anti-actin antibody (Sigma-Aldrich, A2066, 1:4,000 dilution). Primary antibodies were detected with peroxidase AffiniPure F(ab’)2 fragment goat anti mouse IgG (Jackson ImmunoResearch, 115-036-006) at 1:40,000 dilution and goat anti-rabbit IgG (whole molecule)–peroxidase antibody (Sigma-Aldrich, A0545) at 1:40,000 dilution. Blots were imaged and analyzed using the iBright FL1500 Imaging System (Invitrogen).