Fig. 1
H19-GOF mutant exhibits enhanced interaction with DMD. a Protein score of human or mouse H19-interacting proteins identified by LC-MS. b RT-QPCR detection of RIP assay using the indicated antibodies in C2C12 or HSMC. Mean±SEM, n = 3 independent experiments, one-way ANOVA. c Saturation curve determination using His-tagged recombinant DMD (aa. 3046-3685) and biotinylated H19, indicated as a donor/acceptor pair. The concentration of His-tagged recombinant DMD (aa. 3046–3685) is shown. Mean ± SD, n = 3 independent experiments. d In vitro RNA-protein binding followed by dot blot assays using in vitro transcribed biotinylated H19 sense (sen.) or antisense (a.s.) in the presence of indicated recombinant proteins. LINK-A was included as negative control. BRK (breast tumor kinase) is a confirmed binding partner of LINK-A. Bottom panel: annotation for each dot. A1-F8 indicates individual H19 probes (antisense oligos, 30 bp per probe, from 1 to 2340), F9 indicates antisense sequences of LINK-A 471–550, F10 indicates antisense sequences of LINK-A 1251–1330, F11 and F12 are blank, and F13 and F14 are His6-peptide. e EMSA using His-tagged DMD (aa.3046–3685) or Y/F-A mutant and [γ-32P]-labeled human H19 RNA (nt. 1951–1980), Rlof, or Rgof as indicated. f Competition binding assay to determine the Kd of the interaction between His-tagged DMD (aa.3046–3685) and biotinylated-H191951-80. Unlabeled H191951-80 WT or Rgof mutant served as competitors. Mean±SD, n = 3 independent experiments. g Autoradiography (right) or IB detection using the indicated antibodies (left) of CLIP assay in Dmd- or H19-knockout C2C12 cells expressing the indicated constructs. No significance [n.s.], p > 0.05, *, p < 0.05, **, p < 0.01, ***, p < 0.001