Fig. 1. Identification of novel CaMKK2 interaction partners.

A Schematic outlining the proteomics experimental design and workflow to identify CaMKK2-interacting proteins by co-immunoprecipitation (co-IP). HEK293 cells were transfected with EGFP-tagged CaMKK2, co-immunoprecipitated proteins were identified by mass spectrometry and the data were compared with a previously published data set [6]. B Immunoprecipitation of endogenous CaMKK2 from LNCaP cells showed co-immunoprecipitation of the Gemin complex. Immunoprecipitates (IP) with a control IgG were compared with those of CaMKK2 IgG, input lysate and unbound supernatant (SN). Bound proteins were evaluated by western blotting with the indicated antibodies. C A peptide array of full-length Gemin4 divided into 20 amino acid peptides with a 17 amino acid overlap was spotted on two nitrocellulose membranes. The membranes were incubated with either GST-CaMKK2 or GST alone. Bound GST protein was detected with a HRP-conjugated GST antibody. Yellow circles indicate peptides that bound CaMKK2 and had overlapping sequence with neighbouring peptides and red circles denote peptides that bound CaMKK2 but did not contain sequence overlap. A schematic drawing illustrates the sequence location of the highlighted peptides, which were concentrated in the first 370 aa of the Gemin4 sequence. D A peptide array of the N-terminal region of Gemin4 (aa 1–370) using 20 aa peptides with a 19 aa overlap. The peptide array was incubated with GST-CaMKK2 and GST control. The yellow box highlights the region in Gemin4 where GST-CaMKK2 but not GST bound. E Multiple sequence alignment of the CaMKK2-binding motif (CBM; aa 351–359) in Gemin4 identified in the peptide array illustrates sequence conservation across species. F To verify the specificity of the interaction in solution recombinant GST-tagged Gemin4 (aa 1–370) was used in a pulldown assay with LNCaP cell extracts. Equal amounts of control GST and GST-Gemin4 were used in the assay. The CaMKK2 binding was detected by western blotting.