Fig. 3

Impaired phosphorylation enhanced poly-ubiquitination of TET2. a IB analyses of TET2 IP products and WCL derived from HCC827 cells. IgG was used as a negative control. b HCC827 and its derivative cells with knockout of either MEK1 or MEK2 were subjected to IB analysis. c Relative TET2 mRNA level in indicated cells as described in (b). d IB analyses of HCC827^WT cells or HCC827^MEK1-KO cells reintroduced with empty vector, MEK1^WT or MEK1^SA. e CRISPR-Cas9 mediated MEK1 knockout of 293T cells were transfected with indicated constructs and treated with MG132 (10 μM, 12 h) before IP and IB analyses. f IB analyses of HCC827-OR cells without or with the overexpression of constitutive MEK1 (MEK1^CA). g Flow chart to show Flag IP products from 293T^MEK1-KO cells transfected with indicated constructs and treated with MG132 were subjected to phosphoproteomics. h Alignment of putative TET2 phosphorylation residues among different species. The asterisks below indicate the four potential phosphorylated amino acids. i IB analyses of Flag IP products from 293T^MEK1-KO cells treated with MG132 (10 μM, 12 h) as shown in (g). pSer pan phospho-serine, pThr pan phospho-threonine, pTyr pan phospho-tyrosine. j IB analyses of endogenous TET2 immunoprecipitated from PC-9 (up) or HCC827 (bottom) cells with or without osimertinib treatment. k 293T^MEK1-KO cells were transfected with indicated constructs and treated with MG132 (10 μM, 12 h) before harvested for Flag immunoprecipitation, the IP products and WCL were subjected to IB analyses. l 293T^TET2-KO cells were transfected with indicated constructs and treated with MG132 (10 μM, 12 h) before harvested for Flag immunoprecipitation, the IP products and WCL were subjected to IB analyses. All data are representative of at least two independent experiments