Determination of IL-1Ra mRNA levels in HepG2 cells and in human primary hepatocytes by RNase protection assay. HepG2 cells were cultured in the absence (control) or presence of IL-1β , IL-6, and a combination of IL-1β and IL-6 both without (□) and with the addition of 10 ng/mL IL-4 (▩) or 10 ng/mL IL-13 (▩) for 16 hours (A). Ten micrograms of total RNA from the HepG2 cells were hybridized simultaneously with ³²P-labeled riboprobes complementary to IL-1Ra and GAPDH mRNA, then digested with RNase A and RNase T1. The protected fragments were analyzed in a 6% denaturing polyacrylamide gel. The intensity of the protected RNA fragments was analyzed by phosphorImager. The results represent IL-1Ra/GAPDH ratio × 10³. Values represent the mean ± SEM of three experiments. ^*P < .05 in comparison with cells cultured in the absence of IL-4 or IL-13. Human primary hepatocytes were cultured in the absence (control) or presence of IL-1β, IL-6, and a combination of IL-1β and IL-6, both without and with the addition of 10 ng/mL IL-4 or 10 ng/mL IL-13 for 16 hours (B). Ten micrograms of total RNA from the hepatocytes were examined by RNase protection assay as described above. These results are representative of three different experiments.