Figure 5.

A. Summary of phosphopeptides (categorized by phosphorylation motif) found to be enriched in cells expressing wild-type NEK6 under a doxycycline-inducible promoter as compared to kinase-dead NEK6 or cells not treated with doxycycline. B. NEK6 can phosphorylate NCOA5 and FOXJ2 in vitro at the sites discovered in the phosphoproteomic screen. 293T cells were transfected with expression constructs for wild-type and mutant (S-to-D) versions of NCOA5 or FOXJ2 with a C-terminal V5 tag and immunoprecipitated with anti-V5 antibody. Eluates from 1/5 of the beads were assayed by V5 immunoblot; the remaining 4/5 was subjected to on-bead in vitro kinase assay with recombinant active GST-NEK6 (Sigma).