Figure 1. Binding of SD-4 to DC-HIL leads it to associate with syntenin and CD148 on activated T cells.

(A) At varying times after stimulation of human CD4⁺ T cells with anti- CD3 Ab, protein expression of CD148, syntenin or β-actin was examined by immunoblotting. Stimulation index for CD148 protein expression is 1.7-fold increase on day 1; 2.3-fold on day 2; and 5.3 on day 3; and for syntenin, 1.2-fold increase on day 1; 1.6 on day 2; and 2.6 on day 3. (B) Kinetics of CD148 surface expression was also examined by flow cytometry. Expression level is expressed as mean fluorescent intensity (MFI) left after subtracting MFI of control staining from that of positive staining (ΔMFI). (C) At varying incubation times after treatment of PMA/ionomycin-activated CD4⁺ T cells with immobilized control Ig (Ctrl) or DC-HIL-Fc, SD-4 protein was immunoprecipitated from whole T cell extracts and examined by immunoblotting for precipitation of syntenin, CD148 or SD-4. (D) Activated CD4⁺ T cells were treated with DC-HIL-Fc or ICAM1-Fc, fluorescently labeled with anti-SD-4 (shown in green) or anti-CD148 Ab (in red) and co-localization analyzed using confocal microscopy. Among T cells treated with DC-HIL-Fc, 91% (60 of 66 cells examined) showed co-localization with CD148; and among T cells treated with ICAM-1-Fc, 87% (40 of 46 cells examined) did not co-localize. Data shown are representative of at least 2 experiments.