Figure 1.

CD40 ligation translocates and activates SphK1. A) HEK-CD40 cells were transfected with V5-SphK1 and stimulated with agonistic anti-CD40 antibody for the indicated times. Cells were stained with Hoechst (blue) and V5 antibody (green) and visualized by confocal microscopy. Scale bars = 10 μm. B, C) Serum-starved HEK-CD40 cells were stimulated with anti-CD40 antibody for the indicated times in the presence of vehicle or U0126 (1 μM). SphK1 activity (B) and SphK2 activity (C) in cell lysates were measured by formation of S1P. Data are expressed as means ± sd from triplicate determinations. Similar results were obtained in 2 additional experiments. D) In duplicate cultures, phospho-Ser225-SphK1, total SphK1, and pERK1/2 were evaluated by Western blot analysis with the indicated antibodies. One set of representative data is shown from 3 independent experiments. Quantified data by densitometry are expressed as mean ± sd fold vs. time 0; n = 3.