The effect of insulin on PPP1R12B phosphorylation. Immunoprecipitated FLAG-tagged PPP1R12B was resolved by 10% SDS-PAGE and stained with Coomassie blue to visualize the protein. The gel area corresponding to PPP1R12B was excised and subjected to trypsin digestion and HPLC-ESI-MS/MS analysis as described in the Methods section. Relative quantification of each phosphopeptide was obtained by comparing normalized peak-area ratios against the average of 8 representative PPP1R12B peptides. Each PPP1R12B phosphorylation site was normalized by the average value of the respective basal sample and then expressed as fold change over control (n = 4). Box and whisker plots of the ratios were utilized to visualize the effect of insulin on PPP1R12B phosphorylation.