Lipid-Protein Overlay Assay.

This assay was performed as previously described [32]. In brief, C1P and sphingosine spotted onto PVDF membranes. Membranes were dried, blocked with 3% fatty acid free (FAF)-BSA/TBST (0.1% Tween 20) for 2h and incubated with 0.5 μg/ml protein in 3% FAF-BSA/TBST overnight at 4°C. The membranes were washed 5 times for 5 min in TBST and incubated with primary antibodies for 1 hour in 3% FAF-BSA/TBST at room temperature, followed by washing and incubation with the secondary antibody for 1h at room temperature. The membranes were washed 12 times for 5 min in TBST and images were acquired using the Azure c600 imaging system.