Figure S1

Abstract

Gab1 peptide array overlay assay identifies potential binding sites for the PH domain. For this assay, the full amino acid sequence of Gab1 from Mus musculus used in the study of Eulenfeld and Schaper [25] was chemically synthesized as an array of spots of overlapping peptides (Multipep synthesiser [Intavis], with a peptide length of 23 amino acids, sliding two residues further with each consecutive peptide), blocked with 5% nonfat dry milk in TrisHCl buffer (pH 7.5) with 100 mM NaCl and 0.1% Tween 20 added and probed initially with 4 µg/ml GST, followed by incubation with anti-GST, HRP-coupled secondary antibody, and ECL detection. No GST binding was detectable to any of the peptides (top panel). The same membrane was then re-probed with 1 µg/ml of affinity-purified GST-PH domain (bottom panel). Series of dark spots correspond to clusters of nonidentical, overlapping peptides that bind to the GST-PH probe. The red box indicates the Ser552 epitope previously implicated in regulating Gab1 PH domain binding by the work of Eulenfeld and Schaper [25]. Similar results were also obtained when DTT was included in the assay to eliminate potential artefacts from non-specific interactions of Cys residues (unpublished data).
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