Representative extracted ion chromatograms from targeted MS/MS analysis of the SITHLSFGR peptide phosphorylated at both S742 and S747 in cells expressing KD GCK-3 (A) or functional GCK-3 (B). Shading shows the peak areas for the doubly phosphorylated SITHLSFGR peptide. These areas were normalized to the CLH-3b tryptic peptide KILTVEEK and are reported on each plot. In cells expressing functional GCK-3, phosphorylation of SITHLSFGR increases 15-fold. Similar fold changes were obtained in three replicate analyses and with normalization of the peak areas to two other CHL-3b tryptic peptides, LVHGSSGGIFENESR and YVDSQIGTK.