Figure 5

The effect of proteasomal and lysosomal inhibitors on basal and agonist-induced turnover of the hIP. HEK.hIP cells were incubated with (A) MG132 (10 μM) plus cycloheximide (CHX, 20 μg/ml), (B) MG132 (10 μM) for 30 min prior to incubation with cicaprost (1 μM), (C) MG132 (10 μM) plus CHX (20 μg/ml) for 30 min prior to incubation with cicaprost (1 μM), (D) MG132 (10 μM) plus CLQ (100 μM) or (E) MG132 (10 μM) plus CLQ (100 μM) for 30 min prior to incubation with cicaprost (1 μM) for 0-12 hr. A - E, 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 - E, lower panels). The positions of the molecular size markers (kDa) are indicated to the left of A - E. Data are representative of three to five independent experiments. The bar charts show the mean percentage increase or decrease in levels of the mature (46-66 kDa) species of the hIP ± S.E.M. (n = 3-5) where the level of the mature hIP in untreated (0 hr) cells is assigned a value of 100%. *, p < 0.05 and **, p < 0.01 indicates that the mean percentage levels of the mature hIP was significantly increased or decreased following the respective treatment(s) compared to those levels in untreated (0 hr) cells at that time point.