IL-7 stimulation accelerates IL-7Rα degradation, in a lysosome and proteasome-dependent manner. (A-C) Total IL-7Rα expression (surface plus intracellular) was assessed by flow cytometric analysis of fixed and permeabilized HPB-ALL cells, as described in “Flow cytometric analysis.” Analysis of the data was performed as described in Figure 1. *P < .05, ***P < .001 (1-way analysis of variance, with Bonferroni post test). (B) To determine IL-7Rα half-life, cells were treated with the translation inhibitor cycloheximide (CHX). (C) To determine the pathways of degradation of IL-7Rα, cells were pretreated for 1 hour with 50mM NH₄Cl or 25μM lactacystin, and then stimulated or not with 50 ng/mL IL-7 for 1 hour. The geometric mean of fluorescence of the population was determined by flow cytometry, as shown in the representative histogram. *P < .05 (Student t test, 2-tailed). Data in panels A, B, and C represent mean ± SEM from at least 3 independent experiments. (D) Colocalization between IL-7Rα and lysosomes in the presence or absence of IL-7 (1 hour; 50 ng/mL) was performed using α-human IL-7Rα antibody and LAMP-2 as a lysosome marker. The percentage of cells that displayed colocalization is indicated and was determined as in Figure 2. Representative cells of 3 independent experiments are shown.