Fig. 4. PKM2 enhances TGF-β1 signaling by directly interacting with Smad7.
(A) Luciferase assay in 293T cells transfected with the CAGA-luc reporter and shPKM2. (B) Immunoblot analysis in 293T cells transfected with the shPKM2 and treated with TGF-β1. (C) Luciferase assay in 293T cells transfected with the CAGA-luc reporter and Flag-PKM2. (D) Immunoblot analysis in 293T cells transfected with the Flag-PKM2 and Flag-PKM2-K305Q and treated with TGF-β1. (E) Immunoprecipitation with anti-Flag beads in 293T cells expressing Flag-PKM2 and Myc-tagged Smad2, Smad3, Smad4, and Smad7. (F and G) Immunoprecipitation with anti-Flag beads in 293T cells expressing Myc-Smad7 and Flag-PKM2. (H) Flag-tagged PKM2 and His-Smad7 were pulled down using anti-His beads. (I) The interaction of PKM2 with Smad7 was measured by MST. The Kd value was determined with MO.Affinity Analysis Software. (J) Molecular docking showed binding domain of human PKM2 and Smad7. (K) Domains of human Smad7. ND, N-terminal domain, amino acids 1 to 246; MH2, MH2 domain, amino acids 247 to 426. (L) Immunoprecipitation with anti-Flag beads in 293T cells transfected with Flag-PKM2 and different domains of Smad7. (M) Immunoprecipitation with anti-Flag beads in 293T cells transfected with Myc-Smad7 and Flag-PKM2 mutants. (N) Immunoblot analysis in 293T cells transfected with Flag-PKM2 mutants. (O) 293T cells were transfected with Flag-PKM2 and Myc-Smad7, and cytoplasmic and nuclear protein were separated and immunoprecipitated with anti-Flag beads. (P) 293T cells were transfected with Flag-PKM2 and Myc-Smad7 and stained with antibodies against Flag and Myc. Scale bars, 5 μm. Data are represented as the means ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.