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Fig. 7 BCAS2 sequesters β-catenin in the nucleus via its CC domains. (A) Schematics of full length and deletion mutants of BCAS2. (B, C) HEK293T cells were transfected with Flag-β-catenin and indicated deletion mutants of BCAS2. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Eluted proteins were immunoblotted using anti-HA (B) or anti-GFP antibodies (C) for BCAS2 detection. (D) HEK293T cells transfected with the indicated plasmids were treated with 100 ng/ml LiCl for 12 h, and then subjected to luciferase assay (n=3). ns, not significant; **p<0.01 (Student’s t-test). (E, F) Immunofluorescence staining of β-catenin in Tg(gata1:GFP) embryos at 16 hpf. The embryos were injected with 8 ng bcas2 MO and 300 pg of full-length BCAS2 mRNA or ΔCC1-2 mRNA at the one-cell stage. The relative fluorescence intensity of nuclear β-catenin was quantified in (F) (n=5). ns, not significant; **p<0.01; ****p<0.0001 (Student’s t-test). (G) Transcripts of gata1 were evaluated by WISH in bcas2+/Δ14 embryos injected with 300 pg of BCAS2 mRNA or ΔCC1-2 mRNA. Scale bars, 5 μm (E), 100 μm (G).