Mab β actin ac 15

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)]).

The following polyclonal (pAb) and monoclonal (mAb) antibodies were used: mAb-UL97 (clone 1C4/0.2, produced and kindly provided by Dr. T. Lenac/Prof. S. Jonjic, Dept. Histology and Embryology, Univ. Rijeka, Croatia), pAb-UL97 (06-09, kindly provided by Prof. D. M. Coen, Harvard Medical School, Boston, MA, USA), mAb-UL44 (BS 510, kindly provided by Prof. B. Plachter, Univ. Mainz, Mainz, Germany), mAb-pp65 (65-33, kindly provided by Prof. W. J. Britt, UAB, Birmingham, AL, USA), mAb-β-actin (AC-15, Sigma-Aldrich), mAb-cyclin B1 (sc-7393, Santa Cruz; GNS11, Thermo Fisher Scientific, Waltham, MA, USA), pAb-cyclin B1 (sc-752, Santa Cruz), mAb-cyclin T1 (sc-271348, Santa Cruz), pAb-cyclin T1 (sc-10750, Santa Cruz), pAb-Fc (rabbit Fc fragment, Jackson ImmunoResearch Laboratories, Bar Harbor, ME, USA), mAb-Flag (M2, Sigma-Aldrich), pAb-Flag (F7425, Sigma Aldrich), and mAb-HA (Clone 7, Sigma-Aldrich). The following fluorescent dye-conjugated secondary antibodies were applied in immunofluorescence analyses: Alexa 488-conjugated goat anti-rabbit IgG (H+L) and Alexa 555-conjugated goat anti-mouse IgG (H+L) (Life Technologies, Carlsbad, CA, USA).