Figure 1

Identification of the molecular species of the hIP expressed in HEK.hIP cells. A, western blot analysis of whole cell protein (50 μg) from HEK 293 cells (lane 1) or HEK.hIP cells (lanes 2-4) incubated with vehicle (0.001% DMSO, lanes 1, 2 & 4) or tunicamycin (2 μg/ml, lane 3) for 24 hr. Whole cell protein (50 μg) was treated with endoglycosidase H (Endo H, 200 units/50 μg protein; lane 4). B, HEK 293 cells (lane 1) or HEK.hIP cells (lanes 2-5) were incubated in the absence (-) or presence (+) of SCH66336 (5 nM) for 24 hr prior to harvesting. Where indicated, whole cell protein (50 μg) was treated with PNGase F (500 units/50 μg protein, lanes 4 & 5). A &B, reactions were stopped by the addition of SDS-sample Buffer and proteins were resolved by SDS-PAGE, electroblotted onto PVDF membrane and screened with the anti-HA (3F10) antibody. B, Inset, the region highlighted by the dashed-line box in B is shown following long-term chemiluminescence exposure. The arrows between the panel and inset highlight the apparent molecular weights (MW) 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, respectively. The variably glycosylated mature hIP has an apparent MW of ~46-66 kDa. The higher MW species detected in panel B, lanes 4 & 5 may represent dimeric (*) and oligomeric (**) forms of the hIP. The positions of the molecular size markers (kDa) are indicated to the left of the panels. Data are representative of three independent experiments.