FIGURE 5.

Rab17 sumoylation is nucleotide independent, but prenylation dependent. A, WIF-B lysates expressing the N terminally myc-tagged GTP-bound/Q77L or GDP-bound/N132I rab17 mutants were immunoblotted with antibodies against myc. In B, lysates from cells expressing the N terminally FLAG-tagged GTP-bound/Q77L or GDP-bound/N132I rab17 mutants were immunoblotted (IB) with antibodies against FLAG, whereas in C, Clone 9 cells expressing the myc-tagged mutants were blotted with anti-myc antibodies. Arrows are marking the 40- and 25-kDa immunoreactive species. In A, the asterisk is marking a myc cross-reactive band. D, the relative levels of the 40-kDa immunoreactive species was determined by densitometry and calculated as the percent of total rab17. Values represent the average of at least 3 independent experiments ± S.E. E, lysates from cells expressing a C terminally V5-tagged rab17 were immunoblotted with antibodies against V5. Only the 25-kDa band was detected. F, WIF-B cells expressing rab17-V5 were fractionated by differential centrifugation and fractions were immunoblotted with anti-V5 antibodies (top panel) or antibodies specific for APN (middle) or HDAC6 (bottom). PNS, post-nuclear supernatant; nuc, nuclei; cyt, cytosol; mem, total membranes excluding nuclei.