Identification of proteins interacting with the NEK1-C21ORF2 complex

We and others have found that the NEK1 kinase interacts with the C21ORF2 protein. Gel filtration indicated there may be other proteins in the complex. We tried to identify such proteins by immunoprecipitating endogenous NEK1 from cells and subjecting the precipitates to mass spectrometry. We also immunoprecipitated endogenous NEK1 from cells and subjected the precipitates to mass spectrometry. To find the full complement of NEK1 interactors, NEK1 was immunoprecipitated from ARPE-19 parental cells, and NEK1-KO cells were to control for non-specific binding. Five biological replicates were used per cell line, and after trypsinization of the precipitates, peptides were labelled with tandem mass tags (TMT) enabling quantitative mass spectrometric analysis of the 10 samples which were pooled and analysed in parallel. To find the full complement of C21ORF2 interactors, C21ORF2 was immunoprecipitated from ARPE-19 parental cells, and C21ORF2-KO cells were to control for non-specific binding. Five biological replicates were used per cell line, and after trypsinization of the precipitates, peptides were labelled with tandem mass tags (TMT) enabling quantitative mass spectrometric analysis of the 10 samples which were pooled and analysed in parallel.