Mab lamin a c

For immunogold staining of cellular and viral proteins, HFF-UL50 cells were seeded in T75 flasks, which were then either induced with dox or left uninduced, and infected with purified ΔUL50C particles. At 6 d p.i., cells were carefully trypsinized and centrifuged at 106 rcf for 3 min. Pellets were washed with PBS and centrifuged at 68 rcf for 5 min prior to fixation in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 5 h at 4 °C. Specimens were dehydrated serially to 70% ethanol at −20 °C and embedded in resin (LR White; Electron Microscopy Sciences). Ultrathin sections were successively incubated in Tris-buffered saline (TBS), as well as 0.05 M of glycine, 0.5% ovalbumin, and 0.5% fish gelatin. Primary antibodies were diluted in TBS–ovalbumin overnight at 4 °C, and, finally, 10-nm gold-conjugated secondary antibodies (Bio-Cell) were diluted 1:30 in TBS–ovalbumin for 1 h. After rinsing with TBS, the sections were stained with uranyl acetate and examined with a transmission electron microscope (906E; Zeiss Microscopy). Antibodies used for immunogold staining were mAb-lamin A/C (ab108595, Abcam, Cambridge, UK), pAb-lamin A/C pSer22 (ABIN 1532183, Antibodies online, Aachen, Germany), and pAb-UL53 (kindly provided by P. Dal Monte, Bologna, Italy).

Antibodies used in this study are mAb-IE1p72, mAb-MCP, mAb-pp28 (all kindly provided by William Britt, University of Alabama, Birmingham, AL, USA), mAb-UL44 (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-UL50.01, mAb-UL53.01, mAb-UL97.01 (all kindly provided by Stipan Jonjic and Tihana Lenac Rovis, University of Rijeka, Rijeka, Croatia), mAb-β-Actin (A5441, Sigma Aldrich, St. Louis, MO, USA), mab-lamin A/C (ab108595, Abcam, Cambridge, UK), pAb-lamin A/C pSer22 (ABIN1532183, Antibodies online, Aachen, Germany), pAb-Pin1 (10495-1-AP, Proteintech, Rosemont, IL, USA), anti-Cytomegalovirus-Alexa Fluor 488 (IE1/MAB810X, Merck, Darmstadt, Germany), mAb-HA (Clone 7, H9658, Sigma Aldrich), mAb-Flag (F1804, Sigma Aldrich), mAb-emerin (Sc-25284, Santa Cruz, Dallas, TX, USA), anti-mouse Alexa 555 (A-21422, Thermo Fisher Scientific, Waltham, MA, USA) and anti-rabbit Alexa 488 (A-11008, Thermo Fisher Scientific).