Shotgun RIKA nominates PIM-controlled pathways and potential physiological substrates. (A) RIKA workflow to survey the cellular proteome for protein substrates of PIM1. (B) Top 10 pathways nominated by DAVID analysis of 778 PIM substrates identified in K562 cell extracts. P values were calculated for all PIM involved pathways. The top 10, based on P value and the presence of >10 genes in each pathway, were plotted. Distance of each pathway from center reflects the average enrichment (further = more enriched), and the pathway circle area is proportional to the number of pathway proteins. Gene ontology analysis was performed with DAVID database, as described by Huang et al.³⁵ Kinase substrates identified by mass spectrometry were used for gene overrepresentation analysis. An enrichment P value of < .05 was used in the enriched functional annotation to identify significant biological processes associated with identified kinase substrates. (C) Top, autoradiogram of standard RIKA analysis of recombinant putative PIM substrates. Left, PIM2-containing gel; right, control gel without PIM2; bottom, Coomassie blue-stained RIKA gels from which autoradiograms were obtained. Identical results were obtained with PIM1-containing gels. PIM2 has a lower degree of autophosphorylation compared with PIM1, resulting in higher signal-to-noise in a RIKA.