Fig. 5

BUB1 Kinase Activity Was Required to Maintain STAT3 Target Gene mRNA Levels. A. STAT3 was purified alone or incubated with equimolar amounts of purified BUB1 in the absence or presence of 10 μM 2OH-BNPP1 and was then subjected to immunoblotting using the indicated antibodies. B. An in vitro kinase assay was performed using purified BUB1 and the indicated STAT3 protein, and immunoblotting was then performed with the indicated antibodies. C 5637 cell were cotransfected with BUB1 and MYC-tagged STAT3 or mutants, and immunoblotting was then performed. D. 5637 cell were transfected with si control or two distinct BUB1 siRNAs. Lysates were immunoprecipitated with the anti-pS727-STAT3 antibody, and immunoblotting was then performed with the anti-STAT3 antibody. Lysates were also subjected to immunoblotting with the anti-BUB1 antibody. E. The protein levels of BUB1, Ser727-STAT3, STAT3, NFATC2, LHX1 and GAPDH were measured by Western blotting in BUB1wt vector-transfected 5637 cell. F. 5637 cell were transfected with the BUB1wt vector or BUB1 KD vector. qRT–PCR analysis was performed to measure the mRNA levels of STAT3–induced target genes. The data are presented as the means ± SDs. G. The protein levels of BUB1, Ser727-STAT3, STAT3, NFATC2, LHX1 and GAPDH were measured by Western blotting in BUB1wt vector- or BUB1 KD vector-transfected 5637 cell. H. STAT3 luciferase activity in 5637 cell after transfection with BUB1wt and shSTAT3. The signal was quantified, and statistical significance was determined by Student’s t-test (**p < 0.01, ***p < 0.001). I. Chromatin prepared from 5637 cell treated with 2OH-BNPP1 was subjected to ChIP using an anti-STAT3-Ser727 antibody, and qPCR was then performed using primers targeting STAT3 or the control (gene desert) region. *p < 0.05, **p < 0.01. J. Chromatin prepared from 5637 cells treated with siBUB1 was subjected to ChIP using an anti-STAT3-Ser727 antibody. K. Chromatin prepared from 5637 cell treated with BUB1wt or KD vectors was subjected to ChIP using an anti-STAT3-Ser727 antibody. L. Western blot analysis of protein expression in 5637 cell after transfection with STAT3wt and shBUB1. Signals were quantified, and statistical significance was determined by Student’s t-test (**p < 0.01, ***p < 0.001). M. Sphere formation assay in STAT3wt vector- and shBUB1 vector-transfected 5637 cell