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Fig. 5. Constitutive internalization of IL-2Rβ. (A) HEK293Tβ4 cells grown on coverslips were fixed and then incubated with the following combinations of antibodies: anti-EEA1 monoclonal antibody and anti-IL-2Rβ antibody (C20); anti-LAMP1 monoclonal antibody and anti-IL-2Rβ antibody (C20); and anti-Hrs monoclonal antibody and anti-IL-2Rβ antibody (C20). Subsequently, the cells were incubated with fluorescently labeled secondary antibodies. Scale bars: 5 µm. (B) Internalization of IL-2Rβ in HEK293Tβ4 cells. The radioactivity of cell-surface-bound acid-removable fractions (a) and intracellular acid-unremovable fractions (b) was counted. ¹²⁵I-TU11 binding to parental HEK293T cells was 7.4% of that of HEK293Tβ4 cells. The values represent the mean ± s.e.m. of triplicate determinations. Cells were incubated with (IL-2⁺) or without (IL-2^–) IL-2. (C) Kinetics of the endosomal localization of IL-2Rβ. Cells grown on coverslips were incubated with TU11 at 0°C, followed by treatment with a chemical crosslinker. The cells were incubated at 37°C, fixed at the indicated times and incubated with an anti-Hrs monoclonal antibody. Scale bars: 5 µm.

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