Supplemental data

1. Supplemental data A ILG+Dima Dima Cont ILG c-Fos c-Jun GAPDH B c-Fos 150 c-Jun ** 100 Protein Level (% of GAPDH) 50 *** 0 ILG cont Dima ILG+Dima

2. Supplemental Data Materials and Method Western Blot Treated and control cells (3 × 106) were lysed in 300 µl of 50 mM Tris·HCl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol, 10% glycerol, and 0.05% bromophenol blue and sonicated to shear DNA. Samples were then boiled for 5 min, and 20-µl samples were electrophoresed in 12% SDS-polyacrylamide gels and transferred to nitrocellulose paper. The residual binding sites were blocked with 5% nonfat dried milk in TBST (20 mM Tris·HCl, pH 7.6, 137 mM NaCl, 0.05% Tween-20), and membranes were incubated with 2 µg/ml of the specific primary antibody in 3% nonfat dried milk in TBST. Rabbit sera against c-Myc, c-Jun, and c-Fos proteins were purchased from Santa Cruz Biochemicals (Santa Cruz, CA). All subsequent washes were performed in TBST. Reactivity was developed using an anti-rabbit polyclonal antibody linked to horseradish peroxidase and enhanced chemiluminescence reagents SUPEX (Neuronex, Korea) according to the manufacturer's instructions. Figure legend Isoliquiritigenin inhibits H2R-mediated c-Fos / c-Jun expression. (A) U937 cells were preincubated with 10mM isoliquiritigenin (ILG) for 5min, and then stimulated with 10mM dimaprit (Dima) for 2hrs. Western blot anaylsis was processed as described in Materials and Methods. Equal loading was ascertained by GAPDH. (B) Three independent experiments were quantified by densitometry and the density was normalized to control (GAPDH). Statistical significance was determined by a modified t test. A P value less than 0.05 was considered statistically significant. **P < 0.01 (c-Fos level compared with Dima alone) , ***P < 0.001 (c-Jun level compared with Dima alone). The results are the mean ± SEM of assay triplicates.