FIGURE 4.

Rab11-FIP2-(446–511) blocks the transport of NPC1L1 to PM. A, NPC1L1-RFP were co-expressed with EGFP-tagged Rab11-FIP2 and Rab11-FIP2-(446–511) individually 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, EGFP-tagged Rab11-FIP2 and Rab11-FIP2-(446–511) were co-expressed individually with NPC1L1-T7 in CRL-1601 cells. 48 h after transfection, EGFP-tagged Rab11-FIP2 variants were immunoprecipitated (IP) with anti-EGFP antibody-coupled agarose. Immunoblot (IB) analysis was carried out using the indicated antibodies. Results shown are representative of three independent experiments.