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After the membrane was blocked with 5% skim milk TBST (20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% Tween-20), it was incubated with streptavidin (SA) coupled to horseradish peroxidase (HRP) (1:1000, Pierce) and washed three times with TBST for 10 min