Fig. 5

Effects of eaf1/2 deficiency on WNT/β-catenin signaling during fish erythrogenesis. A Protein levels of P-β-catenin ser 552 and β-catenin in eaf1^−/−, eaf2^−/−, and WT embryos at 16 hpf and 24 hpf (A1-A2), respectively, and quantitative analysis of protein level in each sample (A3-A4). B Endogenous WNT/β-catenin signaling activities in eaf1^−/−, eaf2^−/−, and WT embryos at 16 hpf and 24 hpf, respectively. One-cell stage embryos were injected with TopFlash (as a reporter) and TK-renilla (as an internal control) together, and the injected embryos were collected for assays at 16 hpf and 24 hpf, respectively. C Double staining of gata1aDsRed⁺ and β-Catetin, in the control and embryos injected with eaf1-MO or eaf2-MO at 24 hpf (C1-C15), and quantification of β-Catenin immunofluorescence intensities in gata1aDsRed⁺ cells (C16), and C13-C15 show the magnified views of C10-C12, respectively. D Chromatin immunoprecipitation (ChIP) analysis of the binding enrichment of protein TCF4 on the promoter of gene scl (D1) and gene lmo2 (D2) in eaf1^−/− and eaf2^−/− embryonic cells at 24 hpf, respectively, with anti- IgG used as a negative control. E WISH analysis of the expression of gata1a and hbbe3 in eaf1^−/−, eaf2^−/−, WT embryos and the corresponding groups treated with Wnt activator BIO at 24 hpf (E1-E12), and statistical analysis of WISH results (E13, E14). Each experiment was repeated at least three times, with similar results for two or three replicates, and a representative result was shown. Data are mean ± SD. C1-C15, E1-E12, lateral view, anterior to the left. *P < .05, **P < .01, ***P < .001. NS, not significant. Scale bar = 200 μm (E1-E12), 100 μm (C1-C12), and 50 μm (C13-C15)