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Three times washing with TBST (20 mM tris(hydroxymethyl)-aminomethane, 0.15 M NaCl, 0.1% Tween 20 (v/v), pH 7.6) was performed before incubation with secondary antibodies (goat anti-mouse IgG antibodies and goat anti-rabbit IgG antibodies coupled to horseradish peroxidase from DAKO at 1:5000 in Roti-Block) for 1 h at room temperature followed by washes (3 10 min) in TBST

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