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Fig. 4
Net1 promotes Nodal signal downstream of or parallel to Smad2. (A) Overexpression of Net1 promotes CA-ALK5-induced ARE-luciferase expression. At 36 h after transfecting with ARE-luciferase reporter together with increasing amounts of Net1 expression plasmids, HeLa cells were re-transfected with or without CA-ALK5 for 24 h, and then harvested for luciferase assays. *P<0.05; **P<0.01 (Student's t-test). (B) One-cell-stage zebrafish embryos were injected with 3 pg tar* mRNA alone or together with 4 ng net1 MO1, and then harvested for whole-mount in situ hybridization. (C) HeLa cells were co-transfected with the ARE-luciferase reporter and indicated plasmids. At 36 h after transfection, the cells were harvested for luciferase assays. **P<0.01 (Student's t-test). (D) One-cell-stage zebrafish embryos were injected with 25 pg ca-smad2 mRNA alone or together with 4 ng net1 MO1, and then harvested for whole-mount in situ hybridization. (E) Overexpression of Net1 does not influence the subcellular localization of Smad2. HeLa cells transfected with Flag–Net1 were treated with or without TGF-β1 for 1 h, and then immunostained with anti-Flag (green) and anti-Smad2 (red) antibodies. Nuclei were counterstained with DAPI and are shown in blue. Scale bar: 10 μm. (F) Overexpression of Net1 does not affect the phosphorylation of Smad2. HeLa cells were transfected with Flag-tagged wild-type (WT) Net1 or the Net1-L266E mutant. After 36 h of transfection, these cells were treated with TGF-β1 or left untreated for 4 h, and then harvested for western blotting using the indicated antibodies. Results in A and C are mean±s.d. (n=3). In B and D, the percentages (mean±s.d.) of the affected embryos are shown as calculated from three independent biological repeats with ∼20–25 embryos in each group. UIC, uninjected control.