Fig. (1) SPHK2 is a major sphingosine kinase isoform in A20/2J cells. (A) Expression of SPHK1 and SPHK2 in A20/2J and mouse splenic B cells was detected by reverse transcription-PCR. The PCR products with cDNA from A20/2J cells (lanes 1, 2) or B cells (lanes 3, 4) using primers for SPHK1 (lanes 1, 3) or SPHK2 (2, 4) were shown. (B) Homogenates from A20/2J cells (lanes 1, 2) and mouse splenic B cells (lanes 3, 4) were incubated with sphingosine and [γ-³²P]ATP in the presence of 0.25% Triton X-100 (lanes 1, 3) and 0.2 M KCl (lanes 2, 4) as described in “Materials and Methods.” Lipids were extracted and separated on TLC. (C) Homogenates of A20/2J cells were centrifuged at 100,000 x g for 30 min. Supernatant (open circle; 10 μg, open square; 40 µg) and pellet (closed circle 5 µg, closed square; 20 μg) fractions were incubated with sphingosine and [γ-³²P]ATP in the presence of 0.25% Triton X-100 (left panel) or 0.2 M KCl (right panel) as described in “Materials and Methods.” Essentially the same result was obtained in another independent experiment.