Clone g3 245

For p15, p16, Rb and GAPDH Western blots, whole cell Hot-SDS lysates of murine MSCs or osteosarcoma cell lines were collected as described previously¹⁹ . For p19 and Histon H3 western blots, nuclear lysates were made by washing cells twice with cold PBS, followed by the addition of PBS-Triton X (0.5%) for 10 min, while shaking on ice. Cells were centrifuged twice, washed with PBS-Triton X (0.5%) and the pellet was resuspended in Hot-SDS buffer (1% SDS, 10 mM EDTA, 10 mM Tris pH 7.4) containing protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (Roche). Protein concentrations of lysates were determined with the Biorad DC^TM protein assay kit (Bio-rad, Hercules, CA, USA) according to the manufacturer’s protocol, measured with a microplate reader (Infinite M Plex, Tecan Group Ltd.).
Sample loading, blotting and quantification were performed as previously described¹⁹ . Blots were stained for p15 (1:500, Abcam, Cambridge, UK), p16 (1:1000, clone JC8, Immunologic, WellMed BV, Duiven, The Netherlands), p19 (1:5000, clone ab80, Abcam), Rb (1:500, clone G3-245, BD Pharmingen, San Diego, CA, USA), Histon H3 (1:1000, Cell Signalling, Leiden, The Netherlands), or GAPDH (1:3000, Cell Signalling). Blots were developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) using the ChemiDoc Touch Imaging System (Bio-rad).

PRb and phosphoralized Rb (phospho-Rb) expression was detected by using two monoclonal Retinoblastoma antibodies: pRb (a.a. 332–344), clone G3-245, Pharmingen, dilution 1:300 and phospho-Rb, clone D20B12, dilution 1:100. P16 expression was performed with a monoclonal antibody (clone JC8, dilution 1:400) (Santa Cruz). Secondary antibody detection and visualization were done with the EnVision FLEX™ Kit K8008 (DAKO) or K5005 (Dako) according to standard protocols. Expression levels were assessed and scored by three experienced investigators (AzH, MAH, DR). The results of the HPyV7 LTag expression using the 2 t10 antibody have been described previously [6 ]. In the epithelial cells of 17 thymomas (46 %) marked LTag expression was found. The expression of LTag was in good agreement with earlier performed HPyV7-DNA PCR and/or the HPyV7-FISH [7 (link)].