Sc 69 744

G418 reagent was obtained from Sigma-Aldrich (St. Louis, MI, USA). The purchased commercial antibodies in this study are listed as follows: Mouse monoclonal anti-Myc 9E10 antibody (M4439, Sigma), Mouse monoclonal anti-Flag (M2) antibody (F3165, Sigma), Rabbit polyclonal anti-Flag antibody (F7425, Sigma), Rabbit polyclonal anti-HA antibody (H6908, Sigma), Rabbit polyclonal Anti-human PIAS3 antibody (ab58406, Abcam, Cambridge, UK), Rabbit monoclonal Anti-PML Protein antibody (ab179466, Abcam), Rabbit monoclonal anti-UBC9 antibody (ab75854, Abcam), Mouse mAb against HCMV UL44 (sc-69,744, Santa Cruz Biotechnology, Heidelberg), Mouse anti-HCMV UL84 mAb (sc-56,977, Santa Cruz); Mouse anti-His mAb (66,005-1-Ig, Proteintech, Rosemont, USA), Mouse anti-GST mAb (66,001-2-Ig, Proteintech), Mouse anti-GAPDH mAb (60,004-1-Ig, Proteintech); Dylight 488/549/649-conjugated secondary antibodies, used for immunofluorescence, were from Abbkine, USA. In addition, His-tagged UL70 protein was expressed and purified as described before, and the corresponding rabbit polyclonal antibody against HCMV UL70 was then prepared in our laboratory [38 (link),39 (link)].

For Western blot, whole-cell extracts and virions were resuspended in phosphate buffer saline (PBS), then the pellets were harvested and lysed with cell lysis buffer containing protease inhibitor cocktail. Equal amounts of total protein in the cell lysates were mixed with sodium dodecyl sulfate (SDS) buffer and were boiled at 95°C for 10 min. The proteins were separated by electrophoresis in 10% polyacrylamide gels and blotted onto PVDF membranes (catalog number IPVH00010; Merck). Membranes were sequentially probed with primary antibodies and appropriate peroxidase-conjugated secondary antibodies, excited using High-sig ECL Western Blotting substrate (catalog number 180-501; Tanon), detected using a ChemiDoc™ Touch Imaging system (732BR2203; BIO-RAD), and quantified by densitometry using ImageJ software (National Institutes of Health). The following antibodies of LXR alpha (ab176323; Abcam), anti-ex2/3, anti-pp28 (sc-56975; Santa Cruz), anti-pp52 (sc-69744; Santa Cruz), anti-pp65 (sc-52401; Santa Cruz), anti-gB (Mab13513, Abnova), and anti-GAPDH (Ab8245; Abcam) were used as primary antibodies in this study.