Luciferase assay. Luciferase assays were carried out in HEK293T SB 242235 grown in 12 well plates by co-transfecting the Elk1 luciferase reporter system (1.0 μg pFA-Elk1 and 1.0 μg pFR-Luc for each well) or STAT3 luciferase reporter system plus the indicated constructs. After 24-h transfection, the cells were cultured with or without serum for another 24 h, and then the cell lysates were assayed by Dual Luciferase Assay system (Promega) and detected by Top Count (Packard).
[3H]Thymidine incorporation assay. Stable NIH3T3 cell lines were plated onto 24-well plates at 3.0 × 104 cells/well. After 24 h, the cells were starved overnight. [3H]Thymidine (1 μCi/ml) was added into each well 6 h before harvesting. The reaction products were fixed with 5% trichloroacetic acid and solubilizing in 0.5 M NaOH/0.5% SDS prior to scintillation counting.
Western blot analysis. Transfected cells were lysed with gentle rotation in a lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8.0, and 0.5% NP40) in the presence of protease inhibitors. After centrifugation at 4 °C for 20 min at 12,000 rpm, the cell lysates were resolved by SDS–PAGE and the reaction products were subjected to immunoblotting analysis.
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Western blot and immunoprecipitation Liver tissue and subcellular fractions were
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