Figure 5.
Endosome-to-TGN transport of MPR46 is impaired in OCRL-depleted HeLa cells. (A) HEK-293 cells stably expressing HMY-MPR46 were treated with OCRL- or GFP-specific siRNA for 72 h and cultivated in sulfate-free media containing chlorate. Depletion was monitored by immunoblotting with an anti-OCRL antibody, and an anti-guanine nucleotide dissociation inhibitor (GDI) antibody was used as loading control. (B) Cyclohexamide was added to the sulfate-free media containing chlorate prior to the experiment. Chlorate was substituted by [35S]sulfate to measure retrograde transport of MPR46 to the TGN. The amount of sulfated MPR46 was determined by Ni-NTA precipitation and scintillation counting and was normalized to the protein content. For more details, see the Materials and Methods section. The amount of sulfated MPR46 in OCRL-depleted cells is expressed as percentage of control cells. Each bar is the mean ± SD of two independent experiments, each performed in duplicate. **P = 0.01 (two-tailed Student's t-test).