Journal: Journal of Proteome Research
Article Title: Characterization of the Human NEK7 Interactome Suggests Catalytic and Regulatory Properties Distinct from Those of NEK6
doi: 10.1021/pr500437x
Figure Lengend Snippet: In vitro interaction and phosphorylation assay of candidate interactors of human NEK7. (A) Western blotting (WB) analysis from pull-down (PD) of recombinant NEK7 binding to HEK293T (293T) endogenous CC2D1A, TUBB (β-tubulin), MNAT1, NEK9, ANKS1B, SMC1, SMC3, and PLK1 and not to the RARA or Ni-NTA agarose resin (Ni-NTA). ANKS1B and NEK9 do not bind to 6×His-RARA (right panel), and all proteins show no interaction with Ni-NTA agarose resin (Ni-NTA), therefore demonstrating the assay specificity. Molecular weight (kDa) of the proteins is indicated. The pull-down assay results are based on two independent experiments, and phosphorylation assay results are based on three independent experiments. (B) Human Nek7 in vitro autophosphorylation and phosphorylation of proteins NEK9, MNAT1, CC2D1A, and TUBB2B, retrieved in the yeast two-hybrid screens. The arrowheads indicate the positions of the GST-tagged proteins or GST-control, whereas the arrows indicate the position of the 6×His-NEK7 detected in the autoradiography ( 32 P Autorad), WB, or SDS-PAGE. Molecular weight (kDa) of the proteins is indicated. (C) Western blotting analyses shows the NEK7 requirement for the NEK9 majority phosphorylation. Recombinant NEK9 was incubated in the presence (+) or absence (−) of 6×His-NEK7 or 200 μM ATP. The black arrowheads indicate the positions of GST-NEK9(764–976), gray arrowheads indicate the positions of 6×His-NEK7 in the WB, and the asterisk points to the recombinant NEK9 upper mobility bands (∼51 kDa) in the SDS-PAGE (right panel).
Article Snippet: The following antibodies were used for either immunofluorescence staining or Western blotting purposes: goat anti-NEK7 (sc50756), goat (sc50763) or mouse (sc1004) anti-NEK9, goat anti-α-tubulin (TUBA) (sc8035) (all from Santa Cruz Biotechnology); mouse anti-NEK7 (ab68060), rabbit anti-NEK7 (ab96538), goat anti-PLEKHA8 (ab38748), rabbit anti-β-tubulin (TUBB) (ab15568), rabbit anti-RGS2 (ab36561), rabbit anti-MNAT1 (ab65125), rabbit anti-ANKS1B (ab116083), mouse anti-CC2D1A (ab68302), and mouse anti-pericentrin (PCNT) (ab28144) (all from Abcam).
Techniques: In Vitro, Phosphorylation Assay, Western Blot, Recombinant, Binding Assay, Molecular Weight, Pull Down Assay, Autoradiography, SDS Page, Incubation