Mab flag m2

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.

Lysates from transfected cells were boiled with 4× SDS buffer for 10 min at 95 °C. The lysates were then sonicated for 1 min using the QSonica Q700 Sonicator (QSonica, Newton, MA, USA). Next, the samples were separated on 10% SDS polyacrylamide gels and transferred to PVDF membranes (BioRad, Feldkirchen, Grmany), followed by chemiluminescence detection using a FUSION FX7 imaging system (Vilber Lourmat, Eberhardzell, Germany). Following antibodies were used: mAB Flag M2 (Sigma-Aldrich, St. Louis, MO, USA), pAB human STAT2 H190 (Santa Cruz, Dallas, TX, USA), mAB β-Actin AC15 (Sigma-Aldrich, St. Louis, MO, USA), mAB Halo (G921A, Promega, Fitchburg, MA, USA), pAB IE2 (pAB178; [28 (link)]).

RK-13 and HEK293T cells were cultured essentially as described previously (59 (link), 62 (link)). mAb 58-46 (α-EU-nsp2) (14 (link)), which recognizes the N-terminal domain of nsp2, nsp2TF, and nsp2N, was a kind gift from Dr. Ying Fang. mAb-FLAG (M2) was from Sigma.