Effects of LY294002 on IL-2-induced changes of mRNA and protein levels in GCS and acid SMase. KHYG-1 cells were cultured in the absence (IL-2^-) of IL-2 (100 U/mL) for 12 hour or 36 hours, or rescued with IL-2 supplementation for 24 hours after IL-2 deprivation for 12 hours (IL-2^-→+). In the indicated cases, 30 μM LY294002 was added 1 hour before the rescue by IL-2 supplementation. (A) After extraction of total mRNA, Northern blot analysis was performed for GCS, acid SMase, and β-actin mRNAs as described in “Materials and methods.” Each lane contains 7 μg total mRNA. Equal loading was confirmed by mRNA level of β-actin in each lane. (B) The protein levels of GCS and acid SMase were detected by Western blot analysis as described in “Materials and methods.” Equal amounts of loading were confirmed by protein level of β-actin. The ratios to β-actin in GCS and acid SMase at mRNA and protein levels were calculated by NIH image software and shown at the bottom of each panel. The results were representative of 3 independent experiments.