Fig. 3.

The Ad E3 Ub-ligase participates in the ubiquitination and degradation of AAV large Rep. (a) Top panel: immunoblots of extracts taken 36 h following transfection of 293T cells with HIV LTR-driven AAV2 Rep, an HA-tagged ubiquitin allowing extension only on K48, and E4orf6 as indicated and described in the text, either in the presence or absence of MG132 as shown. In addition, either siRNAs directed toward cullin 5 (Cul 5, lanes 7–10), or scrambled control siRNAs (lanes 1–6) were added as described in the text. Ubiquitinated Rep was detected with anti-HA antibody. Bottom panel: pre-IP Western blot of the same samples in the top panel probed with anti-cullin 5 antibody to monitor specific knockdown of cullin 5. Cellular tubulin was detected as a marker for the amount of protein present in each sample. (b) Immunoblot of cell lysates transfected with plasmids expressing Flag-tagged E1b 55 kDa, large Rep or E4orf6 as indicated. Following transfection, lysates were immunoprecipitated under non-denaturing conditions with an anti-E1b 55 kDa antibody, followed by Western blotting with an anti-large Rep antibody 7B73.2. The lower panel shows the same samples probed with an anti-Flag antibody to demonstrate the presence of E1b 55 kDa. A non-specific band was observed in all lanes as denoted by an asterisk.