A Capsid-Encoded PPxY-Motif Facilitates Adenovirus Entry

Wodrich, Harald; Henaff, Daniel; Jammart, Baptist; Segura-Morales, Carolina; Seelmeir, Sigrid; Coux, Olivier; Ruzsics, Zsolt; Wiethoff, Christopher M.; Kremer, Eric J.

Affinity purified rabbit anti-protein VI antibodies were used at a dilution of 1:2000. Other antibodies used for the study were: rabbit polyclonal anti-Nedd4.1 and anti-Nedd4.2 antibodies that were a kind gift of O. Staub (Lausanne, Switzerland) (dilution 1:1000), rabbit polyclonal anti-WWP2 antibody (sc-30052, Santa Cruz Biotechnology) (dilution 1:200), goat polyclonal anti-WWP1 antibody (sc-11893, Santa Cruz Biotechnology) (dilution 1:200), mouse monoclonal anti-AIP4/Itch antibody (sc-28367, Santa Cruz Biotechnology) (dilution 1:100) and mouse monoclonal anti-GFP antibody (Roche) (dilution 1:500). SDS-PAGE was done using 12% poly-acrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in TBS containing 10% of dry-milk and 0.01% of Tween 20 (Sigma), followed by over-night detection of antigens using primary antibodies diluted in TBS containing 10% of dry-milk and 0.01% of Tween 20 (Sigma). Primary antibodies were detected using HRP-conjugated secondary antibodies against rabbit, goat or mouse (Sigma) at a dilution of 1:5000. Specific signals were revealed using the enhanced chemiluminescence detection system (ECL) (PerkinElmer).