Fig. 2.

Representative western blots of validated GST-tagged PKCε substrate peptides. GST-tagged PKCε substrate peptides (7–12 amino acids in length) and alanine controls were subjected to in vitro thiophosphorylation. Each peptide is shown with its site of phosphorylation and corresponding alanine control peptide. A, four of the six putative substrate peptides that were borderline to the cutoff criteria on the peptide array. B, four of the seven putative substrate peptides that were mistakenly absent from the peptide array. A and B, (Top) signal from α-thiophosphate ester antibody representing level of thiophosphorylation for each peptide. (Middle) Signal from α-GST antibody representing total peptide in reaction. (Bottom) Multiplex image of α-thiophosphate ester and α-GST signals. Each validated peptide has a significant increase in thiophosphorylation signal compared with their corresponding alanine control across three experiments as measured by an unpaired two-tailed t test (see supplemental Fig. S2). PKCε, PKC epsilon.