Figure 4

Effect of proteasomal inhibition on the hIP expressed in HEK.hIP cells. A, HEK.hIP cells were incubated with MG132 (10 μM) for 0-12 hr. Whole cell protein (50 μg) was resolved by SDS-PAGE, electroblotted onto PVDF membrane and screened with the anti-HA (3F10) antibody (upper panel). Inset, the region highlighted by the dashed-line box in A is shown following short-term chemiluminescence exposure where the arrows to the right of the Inset indicate the presence of the four predominant bands that accumulate in HEK.hIP cells following proteasomal inhibition with MG132. These bands correspond to 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, respectively. Alternatively, HEK.hIP cells were incubated with (B) MG132 (10 μM) or epoxomicin (0.1 μM) for 12 hr or (C) PD150606 (20 μM) for 0, 6 or 12 hr. B &C, whole cell protein (50 μg) was resolved by SDS-PAGE, electroblotted onto PVDF membrane and screened with the anti-HA (3F10) antibody (upper panels). In all cases, membranes were stripped and reprobed with the anti-HDJ-2 antibody (A - C, lower panels). The positions of the molecular size markers (kDa) are indicated to the left of A - C. Data are representative of three to five independent experiments. The bar chart to the right of A shows the mean percentage increase or decrease in levels of the mature (46-66 kDa) species of the hIP ± S.E.M. (n = 5) where the level of the mature hIP in untreated (0 hr) cells is assigned a value of 100%.