Cultures were harvested at specific stages by centrifugation at 4°C and stored at −80°C. The cell pellets were resuspended in a lysis buffer [SDS (40 mg/ml), 100 mM tris-HCl (pH 8.6), 10 mM EDTA, 5 mM glycerol-2-phosphate, 5 mM sodium fluoride, 1 mM sodium orthovanadate, and complete protease inhibitors (Roche)] and sonicated at least five times for 30 s at 40% amplitude. The cellular debris was pelleted by centrifugation at 13,000g for 30 min, and the crude protein extract was precipitated from the supernatant with methanol and chloroform. Protein pellet was resuspended in a denaturation buffer containing 6 M urea, 2 M thiourea, and 10 mM tris (pH 8.0). Protein concentration was measured using standard Bradford assay (Bio-Rad).