Fig. 5: Mst1 phosphorylates C/EBP-β, thereby promoting the interaction between FoxO1 and C/EBP-β.

a Left: interaction between FoxO1 and C/EBP-β was examined by pull-down assays. Middle: CBB staining of the gel after SDS–PAGE. Right: diagrams of recombinant human C/EBP-β proteins (full-length and partial) used as preys for pull-down assays. DBD: DNA-binding ___domain, LZ: leucine zipper ___domain, TA: transactivation ___domain. b Coimmunoprecipitation assays with cardiomyocyte lysates. Upper: the results of quantitative analyses (n = 4). Lower: representative images of immunoblot analyses. c Coimmunoprecipitation assays with cardiomyocyte lysates. Upper: the results of quantitative analyses (n = 4). Lower: representative images of immunoblot analyses. d Left: in vitro kinase assays were carried out by incubating recombinant Mst1 with recombinant C/EBP-β. Reactions were analyzed by SDS–PAGE followed by autoradiography. Middle: CBB staining of the recombinant protein-loaded gel after SDS–PAGE. Right: alignment of sequences of the leucine zipper ___domain of human/mouse/rat/chicken/Xenopus C/EBP-β. e MS/MS spectrum of a triply charged ion (m/z 702.01) corresponding to the phosphopeptide NLEpTQHKVLELTAENER from 3xFlag-C/EBP-β phosphorylated by Mst1. The y- and b-ion series confirmed the peptide sequence and phosphothreonine localization. f Upper: cardiomyocytes were transduced with Ad-wild-type 3×Flag-tagged C/EBP-β or Ad-3×Flag-tagged C/EBP-β-PR mutant. Ectopically expressed C/EBP-β proteins were immunoprecipitated with an anti-Flag-M2 antibody and detected with an antibody against either C/EBP-β phosphorylated at Thr299 or total C/EBP-β. Lower: cardiomyocytes were transduced with Ad-Mst1, Ad-DN-Mst1, or Ad-LacZ. Expression and phosphorylation of C/EBP-β were examined by immunoblots with specific antibodies. g An assay for C/EBP-β dimerization. Upper: the results of quantitative analyses (n = 4). Lower: representative images of immunoblot analyses. h Cardiomyocytes were transduced with Ad-FoxO1-WT, Ad-FoxO1-PR mutant, or Ad-FoxO1 nuclear localization signal defective mutant (NLSmut) with or without Ad-Mst1. Expression of phospho-C/EBP-β (Thr299), C/EBP-β, Mst1, and FoxO1 was evaluated with immunoblot analyses. Upper: the results of quantitative analyses (n = 4). Lower: Representative images of immunoblot analyses. All experiments were repeated at least three times, with n representing biologically independent replicates. P values were determined by one-way ANOVA followed by Tukey’s multiple comparison test in (b, c, g, h). Data are mean ± SEM. Source data are provided as a Source Data file.