IL-7Rα internalization via clathrin-coated pits and trafficking into early endosomes is required for efficient IL-7–mediated signaling. (A) Analysis of IL-7Rα colocalization with clathrin and EEA-1 was performed by confocal microscopy. HPB-ALL cells were plated on poly-D-lysine–coated coverslips, incubated at 4°C with α-human IL-7Rα unconjugated antibody, and subsequently with a secondary α-mouse IgG-Alexa 633 antibody (red). Cells were then shifted to 37°C in the presence or absence of IL-7 (50 ng/mL) for 5 minutes, fixed, quenched, and permeabilized. Endosomal compartments were detected using FITC-conjugated antibodies for clathrin heavy-chain or EEA-1, both enhanced by α-FITC-Alexa Fluor 488 secondary antibody (green). The 2 bottom images are 3-dimensional projections and include DAPI-stained nuclei. Representative cells of 3 independent experiments are shown. The percentage of cells that displayed colocalization is indicated and was determined by counting the fraction of cells showing at least 1 colocalization event between red and green fluorescence. (B-C) HPB-ALL cells were pretreated or not with 0.5M sucrose (hypertonic media) for 1 hour, to inhibit formation of clathrin-coated pits, and then stimulated with 50 ng/mL IL-7 or left untreated (Unst) for 30 minutes. (B) IL-7Rα surface expression was analyzed by flow cytometry, and relative expression was calculated by normalizing the geometric mean of fluorescence of the cell population in the presence of IL-7 stimulus, divided by the unstimulated condition. Data represent mean ± SEM from 3 independent experiments: *P < .05. (C) Immunobloting was performed and IL-7 mediated signaling assessed by detection of phosphorylated JAK1/3, STAT5a/b, and AKT. ZAP-70 was used as a loading control.