Dot blot assay for lipid-protein binding.

Lipid membrane coating and protein overlay assays were conducted as previously described (50). Ethanol and chloroform-soluble fatty acids were directly loaded onto a hydrophobic PVDF membrane (MilliporeSigma). The membrane was coated with NPD1, TX14, and RvE1. The fatty acid–coated membranes were dried and blocked with 1% BSA. The HEK293 cells were transfected with GPR37 cDNA with a V5 tag (GPR37-V5) or with an empty vector (mock transfection) using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific). Cell lysates were incubated overnight with the coating membrane at 4°C, and the binding was detected by anti-V5–tagged antibody (mouse, 1:1,000, Thermo Fisher Scientific, catalog 46-0705). The blots were further incubated with an HRP-conjugated secondary antibody and developed in ECL solution (Pierce, Thermo Fisher Scientific). The intensity of lipid-protein binding was evaluated using ImageJ software.