FIGURE 7.

Effect of SNX27 on endocytosis and recycling of MRP4. After 48 h of control siRNA or SNX27 siRNA transfection, HEK293 cells were subjected to an endocytosis assay (A and B) or recycling assay (C and D) as described under “Experimental Procedures.” A and B, measurement of the endocytosis of MRP4. A, the internalized sulfo-NHS-SS-biotin-labeled proteins were precipitated with streptavidin-agarose beads and subjected to Western blot analysis. B, quantification of the internalized MRP4 relative to the surface pool. Data were derived from the band corresponding to MRP4 in A. The signal intensity was quantified using Image Gauge software. Internalization at 0 min was normalized to 0%. Each bar represents the mean ± S.E. from three independent experiments. *, p < 0.05. C and D, measurement of the recycling of MRP4. C, the internalized sulfo-NHS-SS-biotin-labeled proteins remaining after incubation at 37 °C for the time indicated were precipitated with streptavidin-agarose beads and subjected to Western blot analysis. D, quantification of recycled MRP4 relative to the endocytosed pool. Data were derived from the band corresponding to MRP4 in C. The signal intensity was quantified using Image Gauge software. The recycling of MRP4 was calculated as described under “Experimental Procedures.” Each bar represents the mean ± S.E. (error bars) from three independent experiments.