Figure 5.

Analysis of the binding of phosphorylated peptides derived from the site I sequence to the Cas SH3 domain. (A) Representative Western blot of peptide competition assays by using GST-CasSH3 and wild-type His-tagged FRNK. Components were incubated in the presence of increasing amounts (1, 10, 25, 50, or 100 μM) of each peptide indicated. His-FRNK was detected by immunoblotting with the mAb 2A7 followed by detection with ¹²⁵I anti-mouse IgG and densitometric analysis. (B) Quantitation and graphical representation of PhosphorImager data with wild-type His-FRNK with 25 or 100 μM peptide competitor. Values are expressed as a fraction of a control reaction performed in the absence of peptide (open bar, normalized to 1). P values were calculated using a paired t test. Statistical differences (P < 0.05) between either the wild-type peptide or the phosphopeptide and the control P→A peptide are indicated by an asterisk (∗) above the appropriate bar. Statistical differences between the wild type and phosphopeptides themselves are indicated by a double asterisk (∗∗) above the bar representing the wild-type peptide. Data points represent mean values from three independent experiments; error bars represent SD from the mean.