Pa5 31650

Cells were lysed with RIPA buffer (0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, 150 mM NaCl, and 10 mM Tris at pH 7.2) containing protease inhibitor cocktail (Roche). Ten micrograms of the total lysate was separated in 10% SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore). Primary antibodies used in this paper are mouse anti-CMV IE1/2 monoclonal antibody (MAB8131, Millipore), mouse anti-CMV pp52 monoclonal antibody (CH16, Santa Cruz Biotechnology), mouse anti-CMV pp28 monoclonal antibody (CH19, Santa Cruz Biotechnology), rabbit anti-ZAP polyclonal antibody (PA5-31650, Invitrogen), mouse anti-TRIM25 monoclonal antibody (BD Biosciences), rabbit anti-T7 tag monoclonal antibody (D9E1X, Cell Signaling Technology), mouse anti-alpha tubulin monoclonal antibody (DM1A, Abcam) and mouse anti-β-Actin monoclonal antibody (Abcam). Blots were probed with primary antibody (1:500–1:5000) diluted in 5% dehydrated milk in Tris Buffered Saline (TBS) and subsequently the HRP-conjugated secondary antibodies (Pierce) at 1:5000. Blots were washed in TBS three times, incubated with chemiluminescent substrate (SuperSignal West Pico; Thermo Scientific) according to the manufacturer’s protocol, and exposed in G:Box (Syngene) for visualization of bands.

Laboratory adapted HCMV strain AD169 infected cells were fixed in 4% paraformaldehyde solution for 20 minutes and then permeabilized in Methanol:Acetone solution (1:1) at -20°C for 7 minutes, and then blocked with 5% human serum in PBS for 30 minutes. Primary and secondary antibodies were diluted with 5% human serum in PBS. Cells were washed with PBS after primary and after secondary antibody incubations. Primary antibodies used in this paper are mouse anti-CMV IE2 monoclonal antibody (12E2, Santa Cruz Biotechnology), mouse anti-CMV IE1/2 monoclonal antibody (MAB8131, Millipore), and rabbit anti-ZAP polyclonal antibody (PA5-31650, Invitrogen) at 1:500. Alexa-fluor-647 conjugated goat anti-mouse or Alexa-fluor-488 conjugated goat-anti-rabbit IgG secondary antibodies were diluted 1:1000. All images were acquired with Zeiss LSM 710 confocal microscope fitted with 63X/1.4 oil-immersion objective lens.