Mab β actin

Detailed steps of western blotting were previously described [60 (link)]. Briefly, the primary apoptosis antibodies (diluted 1:1000) including cleaved caspase-3 (c-Cas 3) rabbit mAb (Cell Signaling Technology, Inc., Danvers, MA, USA) were used. The internal control primary antibody (diluted 1:5000) was mAb-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Following secondary antibody treatment, these protein signals were detected using enhanced chemiluminescence (ECL) substrate (WesternBright™ ECL HRP, Advansta, Menlo Park, CA, USA).
For c-Cas 3-based flow cytometry, cells were fixed with 70% ethanol, washed, and incubated with 1 μg/mL of c-Cas 3 (Asp175) rabbit mAb (Cell Signaling Technology) at 4 °C for overnight. After washing, cells were incubated with a secondary polyclonal antibody conjugated with Alexa Fluor 488 (ThermoFisher Scientific, San Jose, CA, USA) at room temperature for 1 h. Finally, the c-Cas 3 expression was analyzed by Accuri™ C6 flow cytometry. Cas 8 inhibitor Z-IETD-FMK (100 μM, 2 h) or Cas 9 inhibitor Z-LEHD-FMK (100 μM, 2 h) were applied to examine the involvement of Cas 8 and Cas 9 in apoptosis.

For CoIP analysis, 293T cells were seeded in 10 cm dishes with a density of 5 × 10⁶ cells and used for transient transfection with expression plasmids. Then, 2–3 days post-transfection (d p.t.), CoIP was performed as described previously [61 (link)], using mAb-HA (H9658, Sigma-Aldrich, St. Louis, MO, USA). In brief, cells were lysed in 500 μL CoIP buffer (50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM PMSF, 2 μg/mL aprotinin, 2 μg/mL leupeptin and 2 μg/mL pepstatin). Subsequently, total lysates were incubated with antibody-coated (2 μL of tag-specific or control antibodies) Dynabeads^® Protein A (30 μL per sample; Life Technologies, Carlsbad, CA, USA) for 2 h at 4 °C under rotation. The precipitates were washed five times with 1 mL of CoIP buffer. CoIP samples and lysate controls were taken prior to the addition of the CoIP antibody. SDS-PAGE and standard Western blot analysis of cell lysates was performed using equal protein amounts as described previously [62 (link)]. Antibodies used for staining were mAb-HA (H9658, Sigma-Aldrich, St. Louis, MO, USA), pAb-Flag (F7425, Sigma-Aldrich, St. Louis, MO, USA), pAb-FKBP12 (ab2918, Abcam, Cambridge, UK) and mAb-β-actin (A5441, Sigma-Aldrich, St. Louis, MO, USA).

Antibodies used in this study: mAb-HA (Clone 7, H9658, Sigma Aldrich); pAb-HA (Signalway Eurogentec, College Park, MD, USA); mAb-Flag (F1804, Sigma Aldrich); pAb-Flag (F7425, Sigma Aldrich); mAb-Myc (ab9106, Abcam, Cambridge, UK); pAb-Strep (2-1507-001, IBA Lifesciences, Göttingen, Germany); mouse Fc (mouse Fc fragment, 015-000-008, Dianova, Hamburg, Germany); rabbit Fc (rabbit Fc fragment, 011-000-008, Dianova); mAb-Orf27, mAb-Orf24, mAb-UL50.01, mAb-UL97.01 (all kindly provided by Stipan Jonjic and Tihana Lenac Rovis, University of Rijeka, Rijeka, Croatia); mAb-BFRF1 (kindly provided by Alberto Faggioni, Sapienza University of Rome, Rome, Italy); PepAS-M53 (kindly provided by Walter Muranyi, Universitätsklinikum Heidelberg, Heidelberg, Germany); PepAS-M50 (kindly provided by Zsolt Ruzsics, Virology, University of Freiburg, Freiburg, Germany); mAb-β-Actin (A5441, Sigma Aldrich); mAb-mIE1 (Anti-m123/IE1, MCMV, CROMA101, Center for Proteomics, Rijeka, Croatia); pAb-p32 (sc-48795, Santa Cruz Biotechnology, Dallas, TX, USA); anti-mouse Alexa 488 (A-11001, Thermo Fisher Scientific), anti-rabbit Alexa 555 (A-21428, Thermo Fisher Scientific).

A flow cytometer (Guava easyCyte) applying FlowJo software (Becton-Dickinson) was used to measure apoptosis in terms of annexin V (Strong Biotect Corporation, Taipei, Taiwan)/7AAD [27 (link)] and a pancaspase activity assay kit (Abcam, Cambridge, UK) [23 (link)] assays. Apoptosis was also detected by Western blotting applying primary antibodies for the cleaved forms of poly (ADP-ribose) polymerase (c-PARP) and caspase 3 (c-Cas 3) (Cell Signalling Technology Inc., Danvers, MA, USA) (diluted 1:1000) and internal control mAb-β actin (Sigma-Aldrich, St. Louis, MO, USA) as described previously [14 (link)]. Cas 3/7 activity was determined by a Caspase-Glo^® 3/7 Assay (Promega; Madison, WI, USA) based on luminescent detection as described previously [28 (link)].

The following antibodies were used for this study [71 (link)]: mAb-UL97.01 (kindly provided by T. Lenac and S. Jonick; Department of Histology and Embryology, University of Rijeka, Croatia, used for Wb analysis), pAb-UL97 (kindly provided by D. M. Coen, Harvard Medical School, Boston, used for Wb analysis), mAb-β-Actin (A5441, Sigma Aldrich, used for Wb analysis), mAb-Cyclin B1 (sc-245, Santa Cruz, used for Wb analysis), pAb-Cyclin B1 (AF6000, R&D Systems, used for IP and Wb analysis), pAb-Cyclin T1 (ab226851, abcam, used for IP and Wb analysis), pAb-Cyclin H (LS-C331195, LS Bio, used for IP and Wb analysis), and Fc chicken (003-000-008, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA).

Western blot analyses of infected and mock-infected HFFs were performed as previously described [27 (link),66 (link)]. Antibodies specific for CDK9 (sc-484, Santa Cruz Biotechnology, Dallas, TX, USA), mAb-CDK1 (sc-54, Santa Cruz Biotechnology), pAb CDK2 (Sc-163, Santa Cruz Biotechnology), pAb-CDK7 (sc-723, Santa Cruz Biotechnology), and mAb β-actin (A5441, Sigma Aldrich, St. Louis, MO, USA) were used with an appropriate secondary antibody to detect the respective proteins.