FIG. 3.

The levels of K48-linked polyubiquitinated forms of AAV5 small Rep proteins are significantly reduced in the presence of siRNA against cullin 5. Shown is Western blot analysis of AAV5 small Rep protein K48-specific polyubiquitination in 293 cells in the presence of cullin 5 siRNA (lanes 6 to 8) or negative control siRNA (lanes 1 to 5) during transient transfection. Lysates were isolated 36 to 42 h posttransfection with vectors expressing myc-tagged AAV5 small Rep proteins plus a construct expressing E4orf6 and another expressing HA-tagged K48-specific ubiquitin in the presence of MG132 or equivalent amounts of DMSO. Cells were transfected with each of the constructs, except where noted: no Rep (lane 1); no HA-ubiquitin (lane 2); no E4orf6 (lanes 3 and 6); and no MG132 (lanes 4 and 7). (A) Post-IP Western blot following immunoprecipitation with an anti-Myc antibody, with an immunoblot using an anti-Myc (bottom) or anti-HA (top) antibody. The accumulation (or lack thereof) of polyubiquitinated isoforms of AAV5 small Rep proteins is indicated by the presence of high-molecular-weight laddering or a smearing effect. (B) Pre-IP immunoblot of same samples as in panel A with anti-tubulin and anti-cullin 5 antibodies, showing depletion of cullin 5. The bands marked with an asterisk (*) are nonspecific and of unknown origin. Antibody dilutions were as follows: anti-HA (Sigma-Aldrich; #H9658), 1:5,000; anti-cullin 5 (Santa Cruz Biotechnology, Inc.; #sc-13014), 1:500; anti-c-Myc (Santa Cruz Biotechnology, Inc.; #sc-40), 1:500; goat anti-mouse-HRP (Sigma-Aldrich; #A5278); and goat anti-rabbit-HRP (Sigma-Aldrich; #A9169) secondary antibodies, 1:5,000.