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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2016 May 11;15(7):1669–1681. doi: 10.1158/1535-7163.MCT-15-0182

Figure 3. PAXIP1 regulates the phosphorylation of pY15-CDK1.

Figure 3

(A) GST-tagged fragments of WEE1 representing its N-terminal, kinase and C-terminal domains were co-expressed with TAP-tagged PAXIP1 tBRCT C2 in 293FT cells. (B) The kinase domain of WEE1 interacts with PAXIP1 tBRCT C2. TAP-tBRCT C2 fragments were pulled down with streptavidin and blotted for GST and CBP. TFX: transfection. (C) The interaction between tBRCT C2 of PAXIP1 and WEE1 is modulated by phosphatase treatment. TAP-tBRCT C2 was pulled down and treated with phosphatase for 30 min. Treatment with EDTA partially restores binding. Numbers show relative quantitation based on densitometry analysis. (D) In vitro kinase assay with recombinant WEE1, CDK1 and PAXIP1 tBRCT C2. (E) PAXIP1 was knocked down using shRNA in 293FT cells and pY15-CDK1 levels measured 24 h post transfection.