Known pyrophosphorylation substrates NOLC1, TCOF1, IWS1 were overexpressed in IP6K1-/- double KO HEK293T cells, along with active WT or kinase-dead IP6K1 i.e. background high or low in inositol pyrophosphates. the three proteins were pulled down via their SFB-tag and submitted to a pyrophosphoproteomics workflow. This was to demonstrate the dependence of pyrophosphorylation on PP-InsPs.