JCS024455 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The residues 428-466 of Hrs comprise one of the IL-2Rβ-binding regions. (A) Structures of wild-type Hrs and its mutants. The Vps27-Hrs-STAM (VHS), Fab1-YGL023-Vps27-EEA1 (FYVE), coiled-coil and clathrin-binding domain (CBD) are shown at the top. The ubiquitin-interacting motif (UIM), PSAP sequence, proline (Pro)-rich region and proline and/or glutamine (Pro/Gln)-rich region are also indicated. (B) Lysates from 293T cells (2×10⁶) cotransfected with 2 µg IL-2Rβ and 2 µg wild-type Hrs or Hrs mutants were immunoprecipitated with TU11 and immunoblotted with an anti-Hrs monoclonal antibody. The levels of IL-2β and Hrs were examined by immunoblotting with an anti-IL-2Rβ antibody (C-20) and anti-Hrs antibody, respectively, as controls. Total lysate: aliquots (1.25%) of lysates from the indicated cells (2×10⁶) were immunoblotted with an anti-Hrs antibody. WT, wild type; IP, immunoprecipitation; IB, immunoblotting.

• Supplemental Figure S2 -

  Fig. S2. Internalization and degradation of IL-2Rβ in BAF-B03 transfectants treated with IL-2. (A) Internalization of IL-2Rβ in the transfectants. F7 cells were incubated in the presence or absence of human recombinant IL-2 (1nM). Other mutant cells, S25, BAFβ_d349-410-3 and BAFβ_d349-410-4, were incubated with 1nM human IL-2. The radioactivity of cell surface-bound acid-removable fractions (a) and intracellular acid-unremovable fractions (b) was counted. (B) Degradation of IL-2Rβ in the transfectants. Cells were treated as described in A. The radioactivity of culture supernatants (a), cell precipitate fractions (b) and TCA-soluble fractions of culture supernatants (c) was counted. The values represent the mean ± s.e.m. of triplicate determinations. Cells were incubated with (IL-2+) or without (IL-2-) IL-2.

• Supplemental Figure S3 -

  Fig. S3. Kinetics of IL-2Rβ sorting to transferrin receptor-positive compartments without chemical crosslinker. Cells grown on coverslips were incubated with TU11 and an anti-transferrin receptor antibody at 0°C. The cells were then incubated at 37°C, fixed at the indicated times and incubated with fluorescently labeled secondary antibodies. Red, green and yellow areas indicate IL-2Rβ staining, transferrin receptor staining and colocalization of the red and green staining, respectively. Scale bars: 5 µm.