Proteins (50 mg) extracted from cardiac allograft were subjected to electrophoresis on a 12% SDS-PAGE and then transferred onto PVDF membranes. The membranes were then incubated with primary antibodies for Beclin-1, LC3B and GAPDH at 37uC for 2 h, respectively. The blots were visualized using enhanced chemiluminescence (Amersham).