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. 2009 Nov 4;84(2):1206–1211. doi: 10.1128/JVI.01660-09

FIG. 2.

FIG. 2.

AAV5 small Rep proteins exhibit different E4orf6-dependent and -independent patterns of polyubiquitination in 293 cells during transient transfection. (A) Post-IP Western blot analysis of AAV5 small Rep protein K48-specific or K63-specific polyubiquitination in 293 cells in the absence or presence of adenovirus E4orf6 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 either HA-tagged K48-specific (left panel) or HA-tagged K63-specific (right panel) ubiquitin in the absence or 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 (lane 3), or no MG132 (lane 4). (Left panel) Post-IP Western blot of K48-specific polyubiquitination following immunoprecipitation with an anti-Rep antibody, with an immunoblot using an anti-Myc (bottom) or anti-HA (top) antibody. (Right panel) Post-IP Western blot of K63-specific polyubiquitination following immunoprecipitation with an anti-Rep antibody, with an immunoblot using an anti-Myc (bottom) or anti-HA (top) antibody. (B) Post-IP Western blot analysis of AAV5 Rep protein wild-type, K48-specific, and K63-specific polyubiquitination during AAV5-Ad5 coinfection. AAV5 Rep proteins were immunoprecipitated from 293-infected lysates with anti-Rep antibody and subjected to Western anaylsis using the anti-Rep (left panel), anti-K48-specific ubiquitin (center panel), or anti-K63-specific ubiquitin (right panel) antibody. 293 cells were infected with AAV5 alone (lane 1), Ad5 alone (lane 2), or both AAV and Ad5 (lanes 3 and 4) in the absence (lane 3) or presence (lane 4) of MG132. Antibody dilutions were as follows: anti-HA (Sigma-Aldrich; #H9658), 1:5,000; anti-Rep (ARP; #03-61073), 1:200; anti-c-Myc (Santa Cruz Biotechnology, Inc.; #sc-40), 1:500; anti-ubiquitin (Santa Cruz Biotechnology, Inc.; #sc-8017), 1:500; anti-K48- and anti-K63-ubiquitin antibodies, which recognize only K48 or K63 polyubiquitin chains, as described previously (18), 1:400; and goat anti-mouse-HRP (Sigma-Aldrich; #A5278) secondary antibody, 1:5,000.