Pab416

Protein extraction and western blotting was performed using standard procedures. In brief, 30 μg protein per sample was separated by Bolt^® Bis‐Tris 4–12% SDS/PAGE (Thermo Fisher) and blotted onto polyvinylidene fluoride (PVDF) by iBlot^® (Invitrogen, Carlsbad, CA, USA) and blocked by 4% skim milk in 2% Tris‐buffered saline/Tween. Primary antibodies used in this study were as follows: 2A peptide 1 : 2000 ABS31 (Millipore, Billerica, MA, USA), SV40LT 1 : 400 Pab416 (Abcam, Cambridge, UK), and β‐actin 1 : 10 000 AC‐15 (Abcam). Secondary horseradish peroxidase (HRP) antibodies used in this study were as follows: goat anti‐rabbit (AP156P; Millipore) and rabbit anti‐mouse (ab97046; Abcam).

The anti-T-antigen (anti-AgT) mouse monoclonal antibody (PAb416) was purchased from Abcam. The 3B2 monoclonal anti-BKPyV VP1 antibody, the anti-mouse IgG (whole-molecule)–peroxidase antibody produced in rabbit, and the neuraminidase were purchased from Sigma. The Alexa Fluor Plus 488-conjugated goat anti-mouse IgG(H+L) was purchased from ThermoFisher. The monoclonal anti-CD63 antibody (MX-49.129.5), the monoclonal anti-calnexin antibody (AF18), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Santa Cruz Biotechnology. The monoclonal anti-CD81 antibody (5A6) was kindly provided by J. Dubuisson (Center for Infection and Immunity of Lille, France). The polyclonal anti-CD9 antibody (GTX55564) was purchased from GeneTex. The monoclonal anti-GM130 antibody (35/GM130) was purchased from BD Biosciences.

Cell slides and frozen sections in 4% formaldehyde (10% PFA, Polysciences, Eppelheim, Germany) were diluted in PBS for 10 min and permeabilized with 0.2% Triton X-100 (10%, Sigma-Aldrich) for 10 min at room temperature (RT). Fixed cells were blocked with blocking buffer containing milk powder and PBS for 15 min (37 °C). Primary and secondary antibodies were diluted in blocking buffer and incubated at RT for 50 min each. Primary antibodies included the polyclonal human rabbit anti-β-catenin (1:1000; Cell Signaling Technology, Beverly, MA, USA) and the monoclonal mouse anti-Large tumor antibody (1:50; PAb416, Abcam, Cambridge, MA, USA). Secondary antibodies used were the anti-mouse IgG1–Alexa Fluor 647 (1:800; Abcam), anti-rabbit IgG–Alexa Fluor 488 (1:1000; Abcam) and direct staining with Hoechst 33342 dye (1:1000; Abcam). Fluorescence was detected using inverted fluorescence microscope (EVOS XL Core, Thermo Fisher Scientific, USA). All images were processed using ImageJ software (NIH, Bethesda, MD, USA).