Mab cyclin b1

A total of 0.5 × 10⁶ HFF were seeded in 10 cm² dishes and were either treated with 3MB-PP1, MBV or DMSO as a negative control. On the next day, the cells were infected with the respective HCMV strains with different genomes per cell (4 genomes per cell). On different days post-infection, the cells were harvested, adjusted to 1 × 10⁵ cells and lysed in 2× Laemmli sample buffer. Protein samples were separated on 10% SDS-PAGE (Life Technologies, NP0301BOX, Carlsbad, CA, USA) and transferred to PVDF membranes (Immobilon, ISEQ00010). The filters were probed with specific primary antibodies phospho-Rb (Ser807/811) (Cell Signaling, 8516S), Rb (Cell Signaling, 9309S), pp28 (kindly provided by William Britt), GAPDH (Sigma-Aldrich, G8795), pAb-UL97 (kindly provided by D.M. Coen, Harvard Medical School, Boston, MA, USA), mAb-Cyclin B1 (sc-245, Santa Cruz Biotechnology), Fluorescent-conjugated secondary antibodies (Invitrogen, A10043; Licor,926-32212) were used for detection by using the Odyssey Infrared Imager CLx (LI-COR Biotechnology, Lincoln, NE, USA).

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).

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).