Extended Data Figure 3: RBC export assays with NBD-sphingosine and [3-³H]sphingosine and the requirement of acceptors for S1P release in RBC.

a–f, TLC analysis and quantification of NBD-S1P bands isolated from RBCs supernatant and pellets from export assays at indicated BSA concentrations (three biological replicates per genotype, experiments were repeated twice with similar results). g–j, Export of [3-³H]S1P in RBCs using [3-³H]sphingosine as substrate. Fifty million washed WT and KO RBCs were incubated with 5 μM [3-³H]sphingosine in Tyrode-H buffer containing 0.5% BSA. After 2 h of incubation, lipids from medium (g) and cell pellets (h) were extracted and analysed by TLC. i, j, Quantification of S1P bands from g and h, respectively (six biological replicates per genotype). k, S1P levels in WT and KO RBCs without release. WT and KO RBCs were incubated with 2 μM [3-³H]sphingosine (in ethanol) for 1 h without BSA and quantified. l, m, S1P-preloaded RBCs as in k were incubated with Tyrode-H buffer containing 0.5% BSA for 1 h. S1P from supernatant (l) and cell pellets (m) were quantified (three biological replicates per genotype, experiments were repeated three times with similar results). The data showed that S1P release from RBCs is dependent on BSA. n, o, Phospholipid liposomes and 10% fresh milk can be used to capture S1P release from RBCs (three biological replicates per genotype; experiments were repeated twice with similar results). ***P < 0.001. **P < 0.01, two-tailed unpaired t-test. Data are mean and s.d.

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