Skip to main content
. 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 2. BRCT- containing protein PAXIP1 binds WEE1 at its C-terminal tandem BRCT domain.

Figure 2

(A) pY15-CDK1 and WEE1 levels of cell lines after 1 h of AZD1775 treatment. (B) Tandem Affinity Purification (TAP)-tagged PAXIP1 tBRCT constructs expressed in 293FT cells. (C) Endogenous WEE1 interacts with PAXIP1 tBRCT C2. TAP-tagged PAXIP tBRCT C2 or TAP-GFP were pulled down using streptavidin beads. (D) Immunoprecipitation of endogenous PAXIP1 (using two different PAXIP1 antibodies, 369 and 370) to detect binding to endogenous WEE1. Immunoprecipitation of endogenous BRCA1, another tBRCT-containing protein was used as control. (E) AZD1775 does not disrupt the interaction between PAXIP1 tBRCT C2 and WEE1. TAP-tBRCT C2 or a TAP-GFP were ectopically expressed in 293FT cells. Equal amounts of lysates expressing TAP-tBRCT C2 were used to incubate in the presence (1 μM) or absence of AZD1775 for 1 h. Lysates were pulled down with streptavidin beads and blotted against CBP to demonstrate equivalent pull down of the ectopic proteins (bottom panel). Incubation with AZD1775 slightly increased binding of WEE1 to PAXIP1 tBRCT C2 (right panel). (F) Pull downs of TAP-PAXIP1 tBRCT N1, C1 and C2 were performed using streptavidin beads with and immunoblotted for endogenous WEE1.