Pab flag

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).

SDS-PAGE and standard Western blot analysis, based on equalized amounts of the total cell lysates, were performed as described previously in [64 (link)]. The antibodies used for staining were mAb-HA (H9658, Sigma-Aldrich, USA), pAb-Flag (F7425, Sigma-Aldrich), mAb-IE1p72 (P63-27) [65 (link)], and mAb-pp28 (both kindly provided by William J. Britt, Dept. Pediatrics Microbiol., Univ. Alabama, Birmingham, AL, USA), mAb-UL44 (kindly provided by Bodo Plachter, Virology, Univ. Mainz, Germany), and mAb-actin (A5441, Sigma-Aldrich).

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).