FIG. 3.

Ubiquitination of μ2 in T3D^N-, T1L-, or T3D^C-infected cells. CV-1 cells were transfected with 3 μg of pHAUb per 60-mm-diameter dish and 6 h later were infected with T3D^N, T1L, or T3D^C intermediate subvirion particles, as indicated, at an MOI of 50 PFU/cell and then incubated at 37°C. At 18 h p.i., cells were harvested and equilibrated for total protein concentration. Following lysis in denaturating buffer, samples were immunoprecipitated with rabbit anti-μ2 polyclonal serum (α-μ2 IP). Protein A-Sepharose beads alone (BA) were used as a control. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the anti-μ2 serum followed by HRP-conjugated anti-rabbit IgG (α-μ2 Blot) (upper row) or with mouse anti-HA MAb followed by HRP-conjugated anti-mouse IgG (α-HA Blot) (bottom row). Bound HRP conjugates were detected by chemiluminescence. Asterisks, cross-reaction of HRP-conjugated antibodies with IgG and protein A used in immunoprecipitations.