For SDS–PAGE analysis, 200 lL of protein extract were precipitated by adding 800 lL of ice-cold acetone for 20 min. The proteins were collected by centrifugation at 10,000g for 10 min at 4 C, and the resulting pellet was dissolved in 100 lL of SDS sample buffer (0.0625 M Tris–HCl pH 6.8, 10% (v/v) glycerol, 5% (w/v) SDS, 0.004% (w/v) bromophenol blue and 5% (v/v) 2-mercaptoethanol) (Laemmli, 1970). Then, the sample was heated for 5 min at 95 C and an aliquot was loaded on the gel.