Mcpyv lt cm2b4

Formalin-fixed, paraffin-embedded (FFPE) sections from primary MCC tumours were purchased from Origene and analysed as previously described [32 (link)]. Primary antibodies were: FITC-conjugated anti-CK20 (Dako, dilution 1:50), MCPyV LT CM2B4 (Santa Cruz Biotechnology, dilution 1:125) and ADAM 10 and 17 (Abcam, dilution 1:250). An isotype-matched irrelevant antibody was used as a negative control on sections of tissues in parallel, a rabbit polyclonal isotype control antibody (Abcam) was used to match the ADAM 10 primary antibody. Sections were incubated with appropriate secondary antibodies labelled with different fluorochromes (Alexa Fluor 546 IgG2B, 643 IgG2A, Invitrogen, and IgG (H+L)-TRITC, Jackson ImmunoResearch). All slides were mounted with Immuno-Mount and images were captured with a Zeiss LSM880 confocal laser scanning microscope.

Constitutive GFP expression from the TA.shRNA.tet construct was used to compare the growth behavior of double-infected and uninfected cells. TA.shRNA.tet cells were mixed with approximately 20% of untransduced cells, and changes in the frequency of GFP-positive TA.shRNA cells in the absence and presence of 1 µg/mL Doxycyclin (Dox) were recorded by flow cytometry over time.
Immunoblotting. Immunoblotting was performed as previously described [18 (link)]. The primary antibodies used in this study were directed against MCPyV-LT (CM2B4, Santa Cruz, CA, USA) or β-tubulin (TUB 2.1, Sigma-Aldrich, St. Louis, MO, USA).