FIGURE 10.

Blocking proteases and recycling increases the intracellular pool of constitutively internalized c-Myc-SERT. A, reversible biotinylation assay on transiently expressed c-Myc-SERT in CAD cells. c-Myc-SERT was allowed to internalize for 2 h at 37 °C while incubated with no extra treatment, 10 μg/ml leupeptin, 10 μg/ml leupeptin + 200 μm chloroquine, or 10 μg/ml leupeptin + 25 μm monensin, before stripping the surface biotin. Stripping efficiency was 93–96%. Biotinylated protein was analyzed by SDS-PAGE followed by immunoblotting. A representative blot (representative of five independent experiments) is shown in the lower panel, and quantification of the data is shown in the upper panel. The background immunosignal seen in the strip control (ice plus strip) was subtracted from the immunosignal of the internalized samples before normalization. Data are means ± S.E., **, p < 0.01, one-way ANOVA, Bonferroni's multiple comparison test, n = 5. B, pulse-chase biotinylation assay on c-Myc-SERT in HEK293 cells. c-Myc-SERT was biotinylated with a nonreducible biotin and allowed to internalize for 4 h ± 100 μg/ml leupeptin. Remaining biotinylated protein was analyzed by SDS-PAGE followed by immunoblotting. The experiment shown (lower panel) is representative of five independent experiments, and quantification of the data is shown in the upper panel. Quantification of the remaining SERT was normalized to a third sample lysed before internalization. Data are means ± S.E. *, p < 0.05, t test, n = 5.