Protein interactions.

(A) Characterization of TAT-CRIB and TAT-mutCRIB proteins. Features and functional domains of the fusion proteins: The fusion proteins are about 29 kd in size and contain a 6-histidine tag (His6), amino acids 47 to 57 of the HIV-TAT protein, a hemagglutinin tag (HA), and the extended CRIB domain of WASP. Critical residues in the CRIB domain and their substitution to alanine are shown in bold. (B) TAT-CRIB associates specifically with Cdc42-GTP. GST-Cdc42 or GST-Rac (2.5 μg) were coupled on GSH-agarose beads and loaded with GDP or GTP in 0.5 μM MgCl₂ buffer. Purified TAT fusion proteins (100 ng) were added for 1 hour in a phosphate-buffered saline buffer containing 5 mM NH₄2SO₄, 10 mM MgCl₂, 300 mM NaCl, and 0.1 mg/mL bovine serum albumin. Following 3 washes, proteins bound to the beads were recovered, separated by 10% SDS-PAGE, and immunoblotted with the following antibodies: 12CA5 anti-HA–tag to visualize TAT-CRIB, rabbit anti-Cdc42 antiserum, or monoclonal anti-Rac for loading controls.