Mab ie1p72 pul44

Immediately following cell culture supernatant harvest, cells were washed with phosphate buffered saline (PBS) and harvested using 0.25% trypsin. Cells were washed with PBS and lysed with 2X SDS sample buffer (62.5 mM Tris/HCl (pH 6.8), 1 mM EDTA, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.005% bromophenol blue) at 95°C for 10 min followed by a brief vortex. Protein extracts were subjected to SDS-PAGE followed by transfer to a Protran nitrocellulose membrane (Whatman). Immunostaining was performed with the antibodies mAb-β-actin (Ac-15, Sigma), mAb-Flag (M2, Sigma), mAb-IE1p72/pUL44 (Clones DDG9 and CCH2; Dako), mAb-IE2p86 (Santa Cruz), mAb-pp65 (Abcam), mAb-pUL97 (#8, kindly provided by Detlef Michel, Ulm, Germany), mAb-MCP (major capsid protein; 28-4, kindly provided by William Britt, Birmingham, AL, USA) and HRP-conjugated anti-mouse secondary antibody (Pierce). Protein bands were visualised using chemiluminescence. Densitometry of immunostaining was performed using ImageJ software. The mean densitometry values for control infected-cells were assumed to be 100% and this value was used to calculate relative protein expression in HCMV-infected cells treated with HCMV siRNAs with results presented as mean ± SD of duplicate or triplicate biological experiments.

MRC-5 cells were seeded in 6 well plates with underlying coverslips and transfected with siRNAs followed by HCMV infection 24 hours post transfection at an MOI of 0.001 pfu/cell as described above. Immunofluorescence staining was performed at 1, 4 and 7 dpi as previously described [38] (link). Staining was performed with mAb-IE1p72/pUL44 (Clones DDG9 and CCH2; Dako), mAb-pp65 (Abcam), and mAb-gB (Abcam) primary antibodies and Alexa Fluor 488 and 594 goat anti-mouse secondary antibodies. For image analysis, 10 fields of view were captured for each treatment, immunostain and time point at x100 magnification. The percentage of positive pixels in each field of view was then calculated using ImageJ software. The mean positive pixel count for all 10 fields of view in untreated HCMV infected cells was assumed to be 100% and this value used to calculate relative protein expression in HCMV-infected cells treated with HCMV siRNAs. Data presented as mean ± SD.