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. 2014 Mar 20;7(1):19–26. doi: 10.1016/j.celrep.2014.03.013

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© 2014 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

PMC Copyright notice

Identification of Phosphopeptide-Specific Interacting Partners of PIH1D1

(A) FLAG-tagged WT PIH1D1 and K64A mutant immunoprecipitates with RPAP3, RUVBL1, and RUVBL2 subunits of the R2TP complex from HEK293T cells. FLAG-tagged WT PIH1D1, but not the K64A phosphopeptide binding mutant, immunoprecipitates UBR5, RPB1, SNRP116, and TEL2. Proteins were immunoprecipitated from HEK293T cells transiently transfected with FLAG-tagged PIH1D1 proteins or empty vector expressing FLAG.

(B) FLAG-tagged TEL2, SNRP116, and UBR5 untreated with lambda phosphatase bind to recombinant GST-tagged WT PIH1D1 but not K64A mutant. Interactions with GST-tagged WT PIH1D1 are disrupted by lambda phosphatase treatment. FLAG-tagged RPAP3 untreated or treated with lambda phosphatase binds to GST-tagged WT PIH1D1 and K64A mutant.