Lysophospholipid Signaling Pathway Protocol

Lysophosphatidic acid (18:1) and lysophosphatidylcholine (18:1) were purchased from Avanti Polar Lipids (Alabaster, AL). ATX inhibitor BMP22 and specific LPA₂ antagonist compound 35 were synthesized as described in (32 (link)) and (34 (link)). Ki16425 was purchased from Cayman Chemical (Ann Arbor, MI). Stock solutions (10 mM) of BMP22, compound 35, and Ki16425 were prepared in dimethyl sulfoxide (DMSO) for in vitro studies. A stock solution of LPA (1 mM) was prepared as an equimolar complex with charcoal-stripped, fatty acid-free bovine serum albumin (BSA) (Sigma-Aldrich; St. Louis, MO) in phosphate-buffered saline (PBS). Polyethylene glycol 400 (PEG 400) was purchased from Sigma-Aldrich. All cell culture media were purchased from Corning Cellgro (Manassas, VA), and cell culture reagents were from Life Technologies (Grand Island, NY) unless otherwise stated. The fluorescent ATX substrate FS-3 was purchased from Echelon Biosciences (Salt Lake City, UT).