Fig. 5

SF3B6 serves as a DKFZp434J0226 (DKFZ)-binding protein. A RNA pull-down assay performed in the HEK293T cell lysates with biotin-labeled oligos. After pull-down, proteins were subjected to SDS-PAGE and stained by Coomassie brilliant blue. The band indicated by the arrow was subjected to mass spectrometry. B Western blotting analysis determined the specific interaction of sense DKFZ with SF3B6 protein, but not with PCNA protein (negative control). C RNA immunoprecipitation (RIP) of SF3B6 interaction with DKFZ in the HEK293T cells. RNA-protein complexes immunoprecipitated by anti-SF3B6 or control IgG were determined by RT-qPCR using specific primers for DKFZ or MEG3 (negative control). Data are shown as mean ± SD (n = 4); **P < 0.01. D Western blotting analysis of SF3B6 levels in the indicated DKFZ overexpression AsPC-1 cells or DKFZ knockdown MIAPaCa-2 cells. E Western blotting analysis of SF3B6 phosphorylation levels in the indicated DKFZ overexpression AsPC-1 cells or DKFZ knockdown MIAPaCa-2 cells. For SF3B6 phosphorylation detection, cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-SF3B6 antibody, followed by immunoblotting analysis with the anti-phospho-tyrosine antibody