Fig 2: SHP2 inhibition results in reduced pY abundance at its interaction motifs.(A, B) TMT signal-to-noise intensities of GAB1 pY659 (A) and GAB2 pY643 (B) peptides showing dynamic changes in phosphorylation under DMSO- (solid line) and SHP099-treated (dotted line) conditions. (C) MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 nM). Total GAB1, total GAB2, GAB1 pY659, and GAB2 pY643 were analyzed by Western blot with both anti-protein and phosphospecific antibodies as a function of time after EGF addition. Western blot results are representative of at least two independent biological replicates.Figure 4—source data 1.Table showing TMT relative abundance values for Y659 of GAB1.Figure 4—source data 2.Table showing TMT relative abundance values for Y643 of GAB2.

Fig 3: The SH2 domains of SHP2, but not its PTP domain, protect specific GAB1 and GAB2 pY marks.(A) Schematic representation of wild-type, point mutants, and deletion constructs of SHP2 tested. (B, C) Parental-, SHP2 knockout-, or SHP2 knockout U2OS cells stably expressing various SHP2 ‘rescue’ constructs were cultured with or without EGF stimulation (10 nM) for 10 min. Cells were lysed and subjected to Western blotting using anti-pY659-GAB1 and anti-GAB1 antibodies. (D) MDA-MB-468 cells stably expressing wild-type, PTP, or SH2 domains pre-treated with SHP099 were stimulated with EGF and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates.