After three 10-min washes in TPBS (100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 N NaCl, 0.1% Tween-20), membranes were incubated for 30 min at room temperature in the secondary horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, http://www.jacksonimmuno.com).