FIGURE 3.

GDP-locked form of Rab11a (S25N) inhibits the transport of NPC1L1 to the PM. A, EGFP-tagged Rab11a(WT), Rab11a(S20V), and Rab11a(S25N) were co-expressed individually with NPC1L1-RFP in CRL-1601 cells. 48 h after transfection, the cells were either depleted of cholesterol for 60 min (Chol-Dep) or replenished with cholesterol for 60 min (Chol-Rep) following cholesterol depletion. Then the cells were fixed and examined by confocal microscopy. Images show cells that are representative of the whole cell population at each indicated time point. Ctr, control. Bar, 10 μm. B, quantification of the intracellular localization of NPC1L1-RFP shown in A. Error bars represent standard deviations. C, the EGFP-tagged Rab11a was co-expressed individually with NPC1L1-T7 in CRL-1601 cells. 48 h after transfection, cells were collected and immunoprecipitation (IP) performed by incubating cell lysates with anti-EGFP-agarose. Immunoblot (IB) analysis was carried out using the indicated antibodies. D, CRL1601/NPC1L1-EGFP cells were harvested and subjected to IP with control or anti-EGFP agarose. IB analysis was carried out using the indicated antibodies. Results shown are representative of three independent experiments.