Figure 3
Apigenin inhibits TRBP phosphorylation.
(a), Cells were treated with caffeine or apigenin for 24 h. Cell lysates were blotted with anti-phosphorylated Erk and anti-total Erk1/2. Representative results from three independent experiments using Huh7 cells are shown. Similar results were obtained using Hep3B cells. (b), A luciferase assay was performed to determine SRE-driven transcription under apigenin treatment. Caffeine was included as a comparison. Data represent the means ± s.d. from three independent experiments using Huh7 cells. *, p < 0.05 (t-test) compared to the negative control. (c), Huh7 cells were transfected with wild-type TRBP-expressing plasmids followed by treatment with the indicated substances for 24 h. Serine-to-alanine mutant TRBP (SΔA) indicates non-phosphorylated TRBP. Representative results from three independent experiments using Huh7 cells are shown. (d), Substance-treated Huh7 cell lysates were separated using a Mn2+-Phos-tag gel to discriminate the phosphorylated form of TRBP. Representative results from three independent experiments using Huh7 cells are shown. (e), TRBP-expressing Huh7 cells were stably transfected with myc-tagged CA-MEK-expressing plasmids followed by apigenin treatment for 24 h. Phosphorylation status of TRBP was determined by Western blotting. Representative results from three independent experiments are shown. (f), Huh7 cells were stably transfected with myc-tagged CA-MEK-expressing plasmids or myc-tagged DN-Erk-expressing plasmids. The expression of the transfected constructs was confirmed by Western blotting using anti-myc antibodies (left panels). Expression levels of mature miRNAs in those cells with or without apigenin treatment were determined by Northern blotting (right panels). Representative results of at least three independent experiments are shown. Full-length blot images in a, b, c, d, e and f are available in Supplementary Figure 5b, c, d, e and f.
