FIGURE 3.
ACER3 overexpression decreases ULC ceramides while increasing SPH and S1P in cells. A, HSC-1 cells were transiently transfected with pcDNA3 or pcDNA3-ACER3 using Effectene according to the manufacturer's instructions. At 72 h post-transfection, microsomes were prepared from cells. Proteins (80 μg) solubilized from a portion of each microsomal preparation were analyzed by Western blot using an anti-FLAG antibody at a 1:1000 dilution. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, remaining portion of each microsomal preparation from A was measured for alkaline ceramidase activity at pH 9.0 and with the indicated ceramides (150 μm) as substrates. C–E, HSC-1 cells transected with pcDNA3 or pcDNA3-ACER3 were analyzed by ESI/MS/MS for sphingolipids. Sphingolipid levels were normalized to total phospholipids (Pi). Numerical data represent mean values ± S.D. of three independent experiments. Image datum represents one of two independent experiments with similar results. *, p < 0.05 versus control.