Extended Data Figure 6: Heterologous expression of Mfsd2b increased S1P export in cell lines and Mfsd2b is an active exporter for S1P in RBCs.
From: Mfsd2b is essential for the sphingosine-1-phosphate export in erythrocytes and platelets
a, Western blot for expression analysis of mouse SphK1, mMfsd2, hMfsd2b, and hMfsd2b mutants D95A, T157R, and K423A in HEK293 cells. Top panel is probed with anti-Mfsd2b antibody, bottom panel is re-probed with anti-mouse SphK1 antibody. We noted that mouse Mfsd2b protein expressed in HEK293 cells were significantly more glycosylated (arrowheads) compared with human Mfsd2b. This might cause the slightly reduced activity of mMfsd2b compared with hMfsd2b in export assays. b, Localization of human Mfsd2b and corresponding mutants in HEK293 cells. c, d, TLC analysis of S1P from supernatant and cells expressed with mSphK1 (mock) or co-expression of mSphK1 with hMfsd2b, D95A, and T157R mutants. e, Quantification of S1P bands in c. f, Time course of export of S1P from hMfsd2b (WT) and hMfsd2b mutants D95A, K423A in HEK293 cells (three biological replicates for c–f, repeated twice). g, h, Export of S1P is not inhibited by a presence of tenfold excess of S1P in the medium; 20 million RBCs and 1 μM [3-3H]sphingosine were used for export assay (three biological replicates per genotype). ***P < 0.001, one-way ANOVA (f) and two-tailed unpaired t-test (e, g, h). Data are mean and s.d.
