Figure 6

Clarification of the molecular species of the hIP expressed in HEK.hIP cells. A &B, HEK.hIP cells were treated with SCH66336 (5 nM) for 24 hr and then incubated with (A) vehicle (0.001% DMSO) or (B) MG132 (10 μM) for 0-12 hr prior to harvesting. As a control, HEK.hIP cells were treated with MG132 (10 μM) for 12 hr in the absence of SCH66336 (panel to the right of B). Whole cell protein (50 μg) was resolved by SDS-PAGE, electroblotted onto PVDF membrane and screened with the anti-HA (3F10) antibody. Membranes were then stripped and reprobed with the anti-HDJ-2 antibody (lower panels). C, HEK.hIP cells were incubated with vehicle (0.001% DMSO, lanes 1 & 4), MG132 (10 μM, lanes 2 & 5) or SCH66336 (5 nM, lanes 3 & 6) for 12 hr prior to harvesting. Where indicated, whole cell protein (50 μg) was treated with PNGase F (500 units/50 μg protein; lanes 4-6). Inset, the region highlighted by the dashed line box in C is shown following short-term chemiluminescence exposure. D, HEK.hIP cells were pretreated with MG132 (10 μM, lanes 1, 3 & 4) or 10 μM MG132 plus 2 μg/ml tunicamycin (lane 2) for 12 hr prior to harvesting. Thereafter, where indicated, whole cell protein (50 μg) was treated with endoglycosidase H (endo H, 200 units/50 μg protein; lane 3) or PNGase F (500 units/50 μg protein, lane 4), where both short- and long-term exposures are shown. Panels B - D, the respective arrows indicate the presence or absence of the core glycosylated, non-farnesylated (44 kDa); core glycosylated, farnesylated (42 kDa); non-glycosylated, non-farnesylated (40 kDa); and non-glycosylated, farnesylated (38 kDa) species of the hIP detected following the various treatments. The positions of the molecular size markers (kDa) are indicated to the left of the panels. Data are representative of three independent experiments.