Sphk2 regulates SFK expression and activity in MKs. (A) Expression of SPHK2 in human megakaryocytic cell lines, Meg01. The samples were examined using a Leica microscope equipped with ×20 objective lens (NA = 0.5) and a commercial CCD camera. Images were acquired by Axiovision software. Green, SPHK2 or IgG; red, 4′,6-diamidino-2-phenylindole. Scale bar represents 10 µm. All cells examined stained positive for SPHK2. (B) Fold change of Fyn, Lyn, Src, Yes, Fgr, and Hck mRNA by BM-derived mature Sphk2^−/− MKs (white) compared with mature WT MKs (black). The mRNA levels were normalized to Gapdh. Representative of 3 independent experiments performed in triplicate. (C) Western blot analysis of Src and p-SFKs in WT or Sphk2^−/− MKs. Gapdh served as loading control. Representative of 5 independent experiments. (D) Fold change of Src and p-SFKs (418) levels in BM-derived mature Sphk2^−/− MKs (white) compared with mature WT MKs (black). The Src and p-SFKs levels were quantified by western blot. The density of Src or p-SFKs was normalized to Gapdh. Data were pooled from 3 to 4 independent experiments. All error bars represent SEM.