Figure 6.

Representative in-vitro binding assay comparing the binding affinities between GST-tagged Cdc42 wild type (left) and GST-tagged Cdc42(T35A) (right) to PBD46, a minimal GTPase-binding domain peptide for an important Ras regulatory protein, mPAK-3. PBD46 is shown as a doublet as has been shown previously (11). Fractions of purified GST alone were used as non-specific background binding control (data not shown) and did not show any appreciable binding to Cdc42 or Cdc42((T35A), however, any nonspecific binding was subtracted out by the equation:

$$\Delta I=\frac{[(\mathit{Cdc}42\mathit{wt}:\mathit{PBD}-\mathit{GST})-(\mathit{Cdc}42\mathit{wt}:\mathit{GST})}{(\mathit{Cdc}42T35A:\mathit{PBD}-\mathit{GST})-(\mathit{Cdc}42T35A:\mathit{GST})}.$$