Transcriptional overexpression of the sphingosine kinase gene in tumorigenic HS2 cells. (A) Poly A⁺ RNA was isolated from the indicated spleen from diseased spi-1-transgenic mice. Northern blot was carried out using a SPHK1 cDNA probe. As a control of RNA loading, the same filter was stripped and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.²⁸  The hybridized filter was exposed in a phosphorimager (Molecular Dynamics, Bondoufle, France) and the resulting signal quantified using the ImageQuant software package. Values are normalized against GAPDH expression. Basal message level is 1 in 663 HS1 cells. (B) Expression of SPHK1 in HS2 cells. Whole-cell extracts were subjected to Western blot analysis (WB) with antibodies against SPHK1 protein and α-adaptin as loading control. (C) Sphingosine kinase activity of SPHK1 in HS1 and HS2 cells was measured on cell lysates from 663 and 812 HS1 and 606 and 921 HS2 cells as described in “Materials and methods.” Data are means ± SD of duplicated samples in 3 independent experiments.