Fig. 2
TGF-β-treated EMT results in phosphorylation of FoxM1 by PLK1 by direct interaction. a, b A549 (a) and NCI-H460 (b) were treated with TGF-β (5 ng/mL) for 48 h. Immunoprecipitation of cell lysates was performed with normal IgG or anti-PLK1 antibody and then immunoblotting was performed with anti-FoxM1 antibody. c An in vitro kinase assay was performed with an active version of PLK1 with T210D (PLK1-TD), radioactive ATP, and purified GST-FoxM1. GST-tagged TCTP was used as the positive control. d In LC–MS/MS analysis, possible phosphorylation residues of FoxM1 by PLK1 were newly detected at the S25, S360, S361, and S393 sites. e, Purified GST-tagged wild-type, S25A, S361A, S715A, and S25/S361/S715A (AAA) FoxM1 mutants were used for a PLK1 kinase assay with radioactive ATP. f, g Phosphorylation of FoxM1 in A549 (f) and NCI-H358 (g) cells treated with TGF-β for 48 h. Treatment with calf intestinal alkaline phosphatase (CIP) reduced the phosphorylation of FoxM1 and PLK1 in TGF-β-induced EMT. Immunoprecipitation was performed with anti-normal IgG (Fig. S4d-e) or anti-FoxM1 antibody, and then immunoblotting was conducted with anti-p-Serine antibodies. Immunoblotting was performed for FoxM1, PLK1, p-PLK1T210, TCTP, and p-TCTPS46 using specific antibodies. TCTP was used as a positive control of the PLK1 substrate. Data are presented as mean ± SD of at least three independent experiments (significantly different from the experimental control). *p < 0.05; **p < 0.01; ***p < 0.001