FIGURE 7:

Half-life of plasma membrane but not total NHE3 depends on SNX27. (A) Plasma membrane NHE3 protein in control and SNX27 shRNA–expressing cells was biotinylated at 4°C using NHS-SS-biotin. Cells were incubated at 37°C and harvested at various time points. After solubilization, biotinylated protein was recovered with streptavidin-agarose beads and analyzed by Western blotting as described. A single representative blot of three independent experiments with similar results. (B) Densitometric analyses of Western blots. The amount of biotinylated NHE3 protein left at different time periods was normalized to surface NHE3 at time 0 (cells always at 4°C). Error bars represent the SE of four experiments. *p < 0.05 values are comparison between control and SNX27-depleted cells. (C) Total NHE3 levels in control and SNX27- depleted cells treated with cycloheximide (100 μM) over the indicated periods. (D) Densitometric analyses of Western blots were performed to calculate percentage of NHE3 remaining over indicated time periods. A representative blot for four independent experiments is shown. Error bars represent the SE of four experiments.