Fig. 8.

Interaction of HRES-1/Rab4 with T-cell surface and adaptor proteins. A, Direct interaction of the TFR and CD4 with HRES-1/Rab4 in Jurkat cells. 10⁷ Jurkat cells were lysed in 1% NP-40, 10% glycerol, 200 mM NaCl, 5 mM MgCl₂, 50 mM Tris pH 8.0, 1 mM PMSF, 1 mM sodium orthovanadate, 20 mM NaF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin for 30 min at 4°C and the supernatant was obtained by centrifugation at 14,000 × g for 20 min at 4°C. 3 ml of the supernatant was pre-cleared with 1 ml of swollen GSH-agarose beads. 500 µl of pre-cleared supernatant was incubated with 5 µg of HRES-1/Rab4-GST-bound agarose beads or 2.6 µg of GST-bound control beads (~100 pmol of each fusion protein) for 2 h at 4°C with and without 1 mM GTPγS. The beads were pelleted at 500 × g for 5 min at 4°C, washed twice in lysis buffer, resuspended in Laemmli buffer, and analyzed by SDS-PAGE stained with Coomassie brilliant blue (left panel) and by Western blot with the indicated antibodies (right panel). B, Pull-down of CD4, CD2AP, and TCRζ, but not CD8 or Rab5A, from PBL lysates prepared as described for Jurkat cells using agarose beads coupled to HRES-1/Rab4-GST. Antibodies to CD2AP (sc-25272), TCR/CD3ζ (sc-1239), CD8 (sc-53212) and Rab5A (sc-28570) were obtained from Santa Cruz. The results represent four independent experiments.