For immunoprecipitation, frozen cell pellets were lysed in lysis buffer (0.5% NP-40, 50 mM Tris-Cl pH 8.0, 125 mM NaCl, supplemented with protease and phosphatase inhibitors) using an end-over-end rotator at 4uC for 30 minutes. Cell extracts were cleared by centrifugation at 16,0006 g for 15 minutes. Supernatants were incubated with protein A-coated Dynabeads that were coupled to 1 mg anti-HA (HA.11, Covance), 1 mg anti-GFP (3E6, Invitrogen) or 2 mg anti-APC3 (AF3.1, Santa Cruz) mouse monoclonal antibodies at 4uC for 1-2 hours. Beads were washed with PBS and immunoprecipitated protein complexes were eluted by boiling beads in reducing sample buffer for 5 minutes. Cell extracts (pre-IP) were also collected and boiled in reducing sample buffer. For mass spectrometry analysis, protein complexes were resolved by SDS-polyacrylamide gel electrophoresis (Invitrogen) followed by staining with a silver stain kit (Sigma-Aldrich). Protein bands specific to immunoprecipitated pUL21a complex were excised for identification by MS/MS mass spectrometry [47] .