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. 2017 Jul 4;27(9):1083–1099. doi: 10.1038/cr.2017.85

Figure 7.

Figure 7

Smad5 dynamically regulates HK1 activity. (A) GO analysis of 67 potential Smad5 binding proteins identified by IP/MS. (B) HEK293 cells co-expressing of Flag-HK1 and GFP, GFP-Smad5, GFP-Smad1, GFP-Smad8 were immunoprecipitated with the GFP antibody, and specific interaction of GFP-Smad5 with Flag-HK1 is shown by western blotting. (C) HEK293 cells were cotransfected with GFP-Smad5 and Flag-HK1. Extracellular pH was then adjusted to 7.0 or 8.0 for 30 min followed by immunoprecipitation with the anti-Flag antibody. A larger amount of Smad5 interacts with HK1 at pHe 8.0 as compared with that of pHe 7.0. (D) Hexokinase enzyme activity was detected in Smad5 KO and various pre-rescue hES. Data are presented as mean ± SEM of three independent experiments. *P < 0.05. (E) WT and Smad5 KO hES cells were incubated in pH 6.0, pH 7.0 and pH 8.0 buffer for 30 min, respectively, followed by hexokinase enzyme activity detection. Data are presented as mean ± SEM of three independent experiments. *P < 0.05. (F) GFP-Smad5 binding leads to HK1 activity increase in vitro. HK1 activity is measured in vitro after incubation of GFP, GFP-Smad5, GFP-Smad1 or GFP-Smad8 proteins using hexokinase enzyme activity assay kit. Data are presented as mean ± SEM of three independent experiments. *P < 0.05.