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Two hundred micrograms of protein of each sample was collected, suspended in 25 μ L of loading buffer containing 125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% 2-mercaptoethanol, and 0.004% bromophenol blue, completed to 50 μ L with Leammli solution (40 mM Tris, 1% SDS and 1% β -mercaptoethanol)