Fig. 3: Nucleus-translocated GCK acts as a protein kinase to phosphorylate TAZ.

a MCF7 cells were treated with or without hypoxia for 8 h. Cytosolic and nuclear fractions were prepared. IP with an anti-TAZ antibody was performed. b A GST pull-down assay was performed by mixing purified His-TAZ protein with purified GST or GST-GCK in the presence or absence of purified 14-3-3. c An in vitro kinase assay was performed by mixing GST-GCK WT/S398D with His-TAZ WT/T346A proteins in the presence of ATP-γ-S. The samples were then alkylated with PNBM. d MCF7 cells transfected with the indicated plasmids were treated with or without hypoxia for 8 h, after which IP and WB analyses were performed with the indicated antibodies. e Parental MCF7 and BT549 cells and the indicated clones with knocked-in expression of TAZ T346A were stimulated with or without hypoxia for 8 h. IP with an anti-TAZ antibody was performed. f MCF7 cells with or without GCK knockout were treated with or without hypoxia for 8 h. IP with an anti-TAZ antibody was performed. All immunoblotting and immunofluorescence experiments were performed in at least three independent biological replicates, with consistent results observed across repetitions. Representative data are shown. Source data are provided as a Source Data file.