High levels of Flag tagged ubiquitinated species were observed in the absence

These data suggest that the 44 kDa species of the hIP rep resents a precursor protein that may be modified by far nesylation to generate the 42 kDa species. Furthermore, when HEK. hIP cells were pretreated with SCH66336 prior to deglycosylation with PNGase F, only the major ABT-737 Bcl-2 阻害剤 40 kDa, but not the minor 38 kDa, species was detected. Therefore, this 40 kDa species may repre sent the non glycosylated, non farnesylated species of the hIP, while the 38 kDa species, detected only in the absence of SCH66336, may represent the non glyco sylated, farnesylated species of the hIP. It was also notice able that the hIP was detected as higher order oligomeric forms following PNGase F treatment. While these oligomers were detected in the presence or absence of SCH66336, there was a slight decrease in the mobility of each species detected in the presence of SCH66336, suggestive of a lack of farnesylation.

As it is only the fully deglyco sylated, PNGase F treated hIP that forms oligomers, they are most likely to be an artifact of the deglycosylation process itself AEB071 PKC 阻害剤 and hence, are unlikely to be of physiologic or biologic relevance in the cellular setting. Investigation of Turnover of the hIP To examine the turnover of the hIP under basal condi tions, HEK. hIP cells were pretreated with cycloheximide for 0 12 hr to prevent de novo receptor synthesis where vehicle treated cells served as controls. In all cases, hIP expression levels were analysed by western blot analyses, quantified by densitometry and uniform protein loading was further verified by immuno blotting for HDJ 2, a ubiquitously expressed molecular chaperone protein.

No sig nificant turnover of the 46 66 kDa or of the 44 or 42 kDa AG-014699 PF-01367338 species of the hIP was apparent over the 12 hr incubation in vehicle treated cells. Noteworthy, in gen eral, the 42 kDa species of the hIP was poorly detected at the shorter exposure times, but was clearly evident follow ing prolonged exposures of the immunoblots. In the pres ence of CHX, the 44 and 42 kDa species of the hIP were not detected 3 hr post treatment and remained absent upon CHX treatment up to 12 hr. The level of the 46 66 kDa species of the hIP remained stable up to 6 hr following CHX treatment, but from 9 12 hr post treatment there was a minor but significant reduction in levels of this species compared to vehicle treated cells.

The CHX did not appear to be effective at inhibiting protein synthesis beyond 9 hr treatment, such that in the presence of CHX the expression of the hIP appears to recover at 9 12 hr. Hence, consistent with pre vious data, these data suggest that the 44 and 42 kDa species represent newly synthesised species of the hIP and, furthermore, that the 46 66 kDa species represents the mature hIP, which resolved as a broad band character istic of its glycosylation pattern in HEK 293 cells. Overall, these data suggest that the level of hIP expression is stable under basal conditions, with receptor degrada tion being compensated for by de novo receptor synthesis. However, blocking de novo receptor synthesis with the protein synthesis inhibitor CHX revealed minor turnover degradation of the hIP under basal conditions from 9 hr post treatment. It was next sought to determine the effect of agonist stim ulation on the expression and turnover of the hIP.