FIGURE 6.

PMA decreases the binding of Fas agonistic antibody and FasL. A, after aliquots containing 5 × 10⁷ JFSD9 cells (Fas-deficient cells reconstituted with Fas-S peptide) were incubated with 500 ng/ml CH-11 for 90 min in the absence or presence of 20 nm PMA, lysates were prepared as described under “Experimental Procedures.” Half of each lysate was treated with λ-phosphatase (PPase) at 30 °C for 30 min. The Fas DISC was then pulled down with rabbit anti-mouse IgM and protein G-Sepharose as described previously (33). After SDS-PAGE, samples were blotted with mouse monoclonal anti-S-peptide and anti-FADD antibodies. Lower panel, cell lysates. HSP90 served as a loading control. B, after JFSD9 cells were treated for 5 h with the indicated concentration of CH-11 in the absence or presence of 20 nm PMA, apoptotic cells were quantified by annexin-V staining. C, Jurkat cells were treated with diluent (top) or 250 ng/ml CH-11 (bottom) without (gray) or with 20 nm PMA (solid black line) for 90 min at 37 °C, chilled, washed twice with ice-cold PBS, stained with APC-conjugated anti-mouse IgM, fixed, and analyzed by flow cytometry. D, summary of mean fluorescence intensity (MFI) obtained from samples in C and additional samples in the same experiment. E, Jurkat cells were treated with 400 ng/ml FLAG-tagged FasL + 1 μg/ml monoclonal anti-FLAG antibody (M2) for 90 min at 37 °C, chilled, washed twice with ice-cold PBS, stained with Alexa Fluor 647-conjugated anti-mouse IgG on ice for 1 h, fixed, and subjected to flow cytometry. Error bars, ± S.D. of three independent experiments. *, p = 0.001. F, Jurkat cells were treated with the indicated concentration of PMA at 37 °C for 90 min, chilled, washed, incubated with 250 ng/ml CH-11 on ice for 1 h, washed, stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgM on ice for 1 h, washed, fixed, and analyzed by flow cytometry. G, after I2.1 cells were treated with 125 ng/ml CH-11 in the absence or presence of 20 nm PMA for 90 min at 37 °C, bound CH-11 was assayed as detailed in C. Error bars, mean ± S.D. of three independent experiments. **, p = 0.024. Inset, whole cell lysates from Jurkat (lane 1) or I2.1 cells (lane 2) were probed with antibodies to FADD and, as a loading control, HSP90.