Fig. 2. Identification of the COPI coat complex as a novel interaction partner of the CaMKK2-Gemin4 protein complex.

A A schematic outlining the Gemin4 peptide pulldown experiment using biotinylated CaMKK2-binding motif (CBM) as bait from cell lysates of LNCaP cells treated with androgen (R1881) or an ethanol control. Bound proteins were identified by western blotting or mass spectrometry. B The streptavidin pulldown of biotinylated Gemin4 CBM and control peptide was analysed by western blotting to demonstrate enrichment of known interactors CaMKK2 and Gemin proteins. C Proteins that bound specifically to the Gemin4 CBM peptide were identified with mass spectrometry and the data set was assessed by pathway analysis (GO cellular location). The top ranked pathways are shown in a table. D A Gemin4 CBM peptide pulldown was used to validate the novel interactions of Gemin4 identified by the pathway analysis. Proteins bound by the peptide from LNCaP cell extract was analysed by western blot using antibodies for CaMKK2 and three components of the COPI complex; α-COP, δ-COP and ζ-COP.