FIGURE 1.

Validation of monoclonal antibodies (αpS/TmAb^Irs1) generated against MS/MS-identified Irs1 phosphopeptides (19). A, Irs1 Ser(P)/Thr(P) residues (rat, mouse, and human numbering) and their surrounding amino acid sequences (phosphopeptide, phosphorylated residue in red) used for mAb production. The Akt motifs were identified at a medium threshold by GPS 3.0 using rat Irs1 (CAA41264) as the target (91). The ratio of score to cutoff (S/C) is reported. B, the sequence specificity of each αpS/TmAb^Irs1 was verified in an ELISA assay against 15-residue phosphopeptides based upon the seven amino- and carboxyl-terminal residues surrounding the Ser(P)/Thr(P) of interest in rat Irs1. C, CHO cell lines expressing human insulin receptor and either wild type or mutant (A307) rat Irs1 were serum-starved overnight and then stimulated with 30 nm insulin for 30 min. Extracts were blotted with total Irs1 and αpS307^Irs1 antibodies. D, CHO^IR/Irs1 cells were treated with 30 nm insulin for 30 min. Irs1 was immunoblotted with the indicated αpS/TmAb^Irs1; each column represents a separate cell extract, with the same extract being used for each antibody in that column.