TRPCs mediate soluble E-selectin prolongation of PAF-induced Ca²⁺ mobilization. (A) Freshly isolated neutrophils were incubated with pertussis toxin (2 μg/mL) for 1 hour, washed, and then loaded with Fura2-am (2 μM) for 30 minutes at 37°C prior to stimulation with 100 nM PAF. Data shown are expressed as mean area under each curve ± SEM from 3 separate experiments. *Statistically different (P < .05) from PAF/LTB₄- or fMLP-treated controls. n.s. indicates not significant. (B) U73122 (5 μM) and U73343 (5 μM) were added 5 minutes and EGTA (1.25 mM) was added 10 minutes before stimulation with 100 nM PAF. A representative Ca²⁺ trace from 3 separate experiments that were performed is shown. (C) Calcium traces showing the effect of calcium channel inhibitors on the prolonged [Ca²⁺]_i elevation induced by soluble E-selectin. Ruthenium red (RR; 20 nM), MRS1845 (2 μM) were added 5 minutes and Ga³⁺Cl₃ (10 μM) was added 3 minutes before stimulation with 100 nM PAF. The calcium trace shown is representative of 3 separate experiments with similar results. (D) Bar graph representing area under the curves of the graph in panel C, calculated using GraphPad Prism software. Data shown are expressed as mean ± SEM from 3 separate experiments that were performed. *Statistically different (P < .05) from PAF/E-selectin–treated controls.