Figure 5.

Role of SphKs and S1PRs in antigen-induced degranulation of RBL-2H3 cells. (A) RBL-2H3 cells were stimulated with IgE/Ag or ionomycin (1 μM) for 30 min in the absence or presence of pretreatment with 10 μM DMS, and hexosaminidase release was determined. *, P < 0.01 by Student's t test. NS, not significant. (B–D) RBL-2H3 cells were treated with control siRNA or siRNAs specific to SphK1 or SphK2 for 48 h. Cells were lysed, and mRNA and proteins were analyzed by RT-PCR or Western blotting (WB) with SphK-specific antibodies as described in Supplemental Materials and Methods. (C) SphK activity was measured in membrane fractions of IgE-sensitized RBL-2H3 cells after stimulation with Ag for 2 min. (D) In duplicate cultures, degranulation by IgE/Ag (white bars) or ionomycin (diagonally striped bars) was measured. Data are expressed as percent inhibition of degranulation relative to control siRNA. (E and F) RBL-2H3 cells were transfected with S1P₁ or S1P₂ antisense (as), sense (s), or scrambled (scr) oligonucleotides for 48 h and stimulated with IgE/Ag or ionomycin for 60 min, and degranulation was measured. Data are expressed as percent inhibition of degranulation relative to scrambled controls. (F) Representative Western blots showing reduction of S1P₁ and S1P₂ protein expression by antisense oligonucleotides as determined with specific antibodies are shown. Blots were stripped and probed with antitubulin to show equal loading. *, P < 0.05 by Student's t test. NS, not significant.