CD95 Induces a DD-Independent Ca2+ Response
(A) CEM T cells were stimulated with cl-CD95L (100 ng/mL). Cells were lysed, and CD95 was immunoprecipitated. The protein complex was resolved by SDS-PAGE and subjected to immunoblotting. Total lysates served as controls. The column marked “B” indicates treatment with beads alone. Data are representative of three independent experiments.
(B) Left panel: Th17 cells from peripheral blood were stimulated with cl-CD95L (100 ng/mL) for indicated times. PLA was performed with anti-CD95 and anti-PLCγ1 mAbs. Nuclei were stained in blue (DAPI). Red dots were observed when the distance between anti-CD95 and anti-PLCγ1 mAbs was close (≈16 nm). Right panels: Red dots were counted in 200 cells taken from different fields. Data represent means ± SD of three independent experiments.
(C) Schematic diagram of CD95 constructs.
(D) CEM-IRC cells expressing GFP alone or the GFP-fused CD95 constructs shown in (C) were loaded with the Ca2+ probe, Fluo2-AM (1 μM). Cells were stimulated with cl-CD95L (100 ng/mL; arrow), and the intracellular calcium concentration ([Ca2+]i) was monitored. Data are given as means ± SD of three experiments performed independently on n = 20 cells.
(E) HEK cells transfected with the indicated constructs were stimulated with CD95L (100 ng/mL) for indicated times. The CD95 protein complex was immunoprecipitated from cell lysates and subjected to immunoblotting, as indicated. Total lysates served as controls. The column marked “B” indicates treatment with beads alone. Data are representative of three independent experiments.
(F) Activated PBLs were pre-incubated for 1 hr with TAT-control or TAT-CID (10 μM) and stimulated with cl-CD95L (100 ng/mL) for the indicated times. The CD95 protein complex was immunoprecipitated from cell lysates and subjected to immunoblotting, as indicated. Total lysates served as controls. The column marked “B” indicates a treatment with beads alone. Data are representative of three independent experiments.
(G) Activated PBLs from healthy donors were loaded with FuraPE3-AM (1 μM) and pre-treated for 1 hr with TAT-control or TAT-CID (10 μM). Cells were stimulated with cl-CD95L (100 ng/mL; arrow). Data represent means ± SD.