Sphingosine 1 phosphate (s1p)

Monoclonal anti-YAP, monoclonal anti-cyclin D1, polyclonal anti-cleaved caspase 3 and polyclonal anti-pYAP antibodies were obtained from Cell Signalling Technology (Danvers, MA). Polyclonal anti-CCN1 (Cyr61), anti-CCN2 (CTGF) and monoclonal anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA). S1P was obtained from Avanti Polar Lipids (Alabaster, AL). Verteporfin (VP) was obtained from VWR International (Randor, PA). For the VP experiments, cells were treated with 10 μm of VP for 10 min, washed and then treated with 300 nM of S1P for additional 2 h. Cycloheximide (#1041) was obtained from BioVision (Mountain View, CA).

To measure SmAP activity in living parasites, approximately 1,000 schistosomula or individual adult male or female worms (in replicate) were incubated in assay buffer [50 mM Tris–HCl (pH 9), 5 mM KCl, 135 mM NaCl, 10 mM glucose, 10 mM MgCl₂] containing the substrate p-nitrophenyl phosphate (p-NPP, routinely at 2 mM) or nucleoside monophosphate (AMP, CMP, GMP, TMP, 0–2 mM) or sphingosine 1 phosphate (S1P, 0.5 mM) (16 (link)). S1P, obtained from Sigma-Aldrich, was prepared in methanol at 2.6 mM solution as recommended by the manufacturer. In some preparations, S1P was dissolved in 95% methanol, dried under nitrogen gas, and reconstituted in fatty acid-free bovine serum albumin (4 mg/ml). rSmAP was used at 0.5–5 μg/assay, as indicated, and with 0.5 mM S1P. To monitor p-nitrophenol generated following p-NPP substrate cleavage, changes in optical density at 405 nm over time were measured with a Synergy HT spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). Phosphate generated following substrate (nucleoside monophosphate or S1P) cleavage was measured using a Phosphate Colorimetric Assay Kit (BioVision), following the instructions of the manufacturer. Samples were recovered at selected time points from each reaction and substrate cleavage (phosphate generation) was monitored.