Figure 11

Ubiquitination of the hIP. A, HEK 293 (lanes 1 & 2) and HEK.hIP (hIP, lanes 3-6) cells were transiently transfected with pCMV5:3xFlag Ubiquitin (3xFlagUb), pcDNA3:HADynamin^K44A (Dynamin^K44A) or, as controls, with the empty vectors pCMV5 or pcDNA3. B, HEK.hIP (hIP) cells were transiently transfected with pCMV5:3xFlag Ubiquitin (3xFlagUb) or, as control, with pCMV5 empty vector. Some 44 hr later, cells were treated with MG132 (10 μM) for 4 hr (lanes 1 & 3). Additionally, cells were pretreated with MG132 (10 μM) for 30 min prior to incubation with cicaprost (1 μM) for 4 hr (lane 2). As a control, cells were treated with cicaprost (1 μM) for 4 hr in the absence of MG132 (lane 4). A &B, thereafter, cells were harvested, lysed in Radio-immunoprecipitation (RIP) Buffer and HA-tagged hIPs were immunoprecipitated (IP) with the anti-HA antibody (IP: anti-HA (Y-11)). Immunoprecipitates were resolved by SDS-PAGE, followed by electroblotting to PVDF membrane. A &B, membranes were initially immunoblotted (IB) with the anti-HA peroxidase-conjugated antibody (IB: anti-HA (3F10POD), lower panels) to detect the HA-tagged hIP and were rescreened with the anti-Flag monoclonal antibody (IB: anti-Flag (Flag M2), upper panel) to detect Flag-tagged ubiquitinated species. The positions of the molecular size markers (kDa) are indicated to the left of A &B. Data are representative of three independent experiments.