2 m naoh

The frozen cell pellets prepared above were resuspended in 3 ml of 2 M NaOH (Fisher Scientific, Waltham, MA, USA) with 10% (vol/vol) beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). The solution was gently mixed and incubated on ice for 5 min to promote hydrolysis of the fungal cell wall. The solution was then centrifuged at 14,000 × g for 30 s at 4°C. The resulting pellet was resuspended in 3 ml of high-salt extraction buffer containing 40 mM HEPES, pH 7.5 (Fisher Scientific, Waltham, MA, USA), 350 mM NaCl (Fisher Scientific, Waltham, MA, USA), 0.1% (wt/vol) Tween 20 (Bio-Rad, Hercules, CA, USA), and 10% (vol/vol) glycerol (Fisher Scientific, Waltham, MA, USA). The solution was immediately centrifuged at 14,000 × g for 30 s at 4°C. The cell pellets were resuspended in 3 ml of 2× SDS sample buffer, 0.1 M Tris-HCl, pH 6.8 (Fisher Scientific, Waltham MA, USA), 4% (wt/vol) SDS (Fisher Scientific, Waltham, MA, USA), 0.2% (wt/vol) bromophenol blue (Sigma-Aldrich, St. Louis, MO, USA), 20% (vol/vol) glycerol (Fisher Scientific, Waltham, MA, USA), and 10% (vol/vol) beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Cell pellets incubated at 100°C for 10 min, prior to being centrifuged at 14,000 × g for 30 s at 4°C. The supernatants were stored at −20°C until further use.