Figure 10

Immunodetection of ubiquitination of the hIP. A, HEK 293 (lanes 1-4) and HEK.hIP (hIP, lanes 5-8) cells were incubated in the presence (+) or absence (-) of vehicle (0.001% DMSO), cicaprost (1 μM) or MG132 (10 μM) for 4 hr. Additionally, cells were pretreated with MG132 (10 μM) for 30 min prior to incubation with cicaprost (1 μM) for 4 hr. B, HEK 293 (lanes 1 & 2) and HEK.hIP (hIP, lanes 3 & 4) cells were incubated in the presence (+) or absence (-) of MG132 (10 μM) for 4 hr. A &B, thereafter, cells were harvested, lysed in Radio-immunoprecipitation (RIP) Buffer and HA-tagged hIPs were immunoprecipitated (IP) using the anti-HA antibody (IP: anti-HA (Y-11)), as described under Experimental Procedures. Immunoprecipitates were resolved by SDS-PAGE, followed by electroblotting to PVDF membrane. In each case, membranes were initially immunoblotted (IB) with the anti-HA peroxidase-conjugated antibody (IB: anti-HA (3F10POD), lower panels) to detect the HA-tagged hIP. A, membranes were rescreened with the anti-ubiquitin monoclonal antibody (IB: anti-Ub (P4D1), upper panel), which recognises both mono- and polyubiquitinated proteins. B, membranes were rescreened with the anti-ubiquitin monoclonal antibody (IB: anti-Ub (FK1), upper panel), which recognises polyubiquitinated proteins only. The positions of the molecular size markers (kDa) are indicated to the left of A &B. Data are representative of three independent experiments.