Effect of alcohol on SphK2 expression in hepatocytes and macrophages. Mouse primary hepatocytes were treated with alcohol (0, 50, or 100 mM) for 6 hours. (A) Total RNA was isolated, and the mRNA level of SphK2 was determined using quantitative RT-PCR and normalized to Hprt1 as an internal control. (B) Total protein lysates were prepared, and the protein level of SPHK2 was determined by Western Blot analysis. Relative protein levels were determined by normalizing to loading control ACTIN. (C) RAW 264.7 cells were treated with alcohol (0, 25 or 100 mM) for 48 hours. Total cellular RNA was isolated. Relative mRNA levels of SphK2, S1pr2, Tnfα, Il-1β were determined using quantitative RT-PCR and normalized to Hprt1 as an internal control. (D) RAW 264.7 cells were treated with alcohol (0, 50 or 100 mM) for 6 hours and total cellular protein was isolated. Relative protein levels of SPHK2 were determined and normalized to ACTIN. Results are represented as mean ± SE from each group (n = 3). Statistical significance relative to the no treatment group, *P < 0.05; **P < 0.01.