HCE cells plated on 100-mm dishes coated with fibronectin plus BSA or with BSA alone were incubated for 45 minutes or HCE cells transfected with Rac1 plasmids and then lysed in 0.5 mL of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 15 minutes, and the resultant supernatants (20 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to 5% skim milk for 1 hour at room temperature before incubation for 1 hour with antibodies to αPAK, the phospho-Ser141 of PAK1/2/3, Rac1, myc, and GFP at a 1:500 dilution in washing buffer (20 mM Tris-HCl [pH 7.4], 5% skim milk, 0.1% Tween 20). The membrane was washed in washing buffer, incubated for 1 hour at room temperature with horseradish peroxidase–conjugated goat secondary antibodies (1:1000 dilution in washing buffer), washed again, incubated with chemiluminescent detection reagents for 5 minutes, and then exposed to film.