Nek6 interacts with and phosphorylates Hsp72. (A) KESTREL analysis of HEK 293 cytosolic extracts. Substrate-containing Superdex 200 fractions 3–7 were pooled, incubated with or without Nek6 and γ-[³²P]ATP, and analyzed by Coomassie blue stain (CB) and autoradiography (³²P). (B) Flag IPs were prepared from HEK 293 cells transfected with Flag-Nek6 and synchronized in S or M. As controls, asynchronous cells (As) transfected with Flag-Nek6 were subject to IP with rabbit IgGs or no antibodies (Ab). IPs were incubated with γ-[³²P]ATP before analysis by Coomassie blue stain and autoradiography (³²P). Lines have been added to top blots to indicate where intervening lanes were spliced out for presentation purposes. In A and B, molecular masses (kilodaltons) and proteins identified by mass spectrometry are indicated. (C) HEK 293 cells were transfected with wild-type (WT) or catalytically inactive (K75M and K64M) Flag-Nek6 or -Nek7 for 24 h before synchronization in S or M. Flag IPs were analyzed by Western blotting with the antibodies indicated. (D) Lysates (inputs), Hsp72 IPs, and IPs prepared with rabbit IgGs from asynchronous, S-phase, or M-phase arrested HEK 293 cells were analyzed by Western blotting with the antibodies indicated. (E) Schematic representation of Nek7, Nek6, and Nek6-ΔNTE. N, N terminus; C, C terminus. (F) In vitro kinase assays using purified wild-type Nek6, Nek6-ΔNTE, or no kinase with β-casein and Hsp72 protein substrates in the presence of γ-[³²P]ATP. (G) Substrate phosphorylation was quantified by scintillation counting of dried gels shown in F. Error bars show means ± SD.