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Figure 4 Deoxyribonucleic acid damage responses in ischemic and reperfused hearts. Representative Western blots of caspase 3 (A), p53, p21WAF, and phosphoH2Ax levels (B), with relative densitometric evaluation and statistical analysis. All experiments were repeated at least 3 x, and differences observed were not due to different protein concentrations as all membranes were also probed with anti-extracellular signal-regulated kinase-1/2 antibodies (B, lower panel). For p53, p21WAF, and phospho-H2Ax, *p < 0.01 vs. control (CON). Molecular weight markers are reported on the left of each panel. (C) Proliferative cell nuclear antigen (PCNA) staining of control, ischemic (58'), and reperfused ventricular samples. Hystochemical staining with anti-PCNA monoclonal antibody was performed as described in the Methods section. Positive cells were counted in at least five (20 x) fields (control samples <0.5 ± 1.2%; ischemia 58 min: 10 ± 3%; reperfusion samples: 8 ± 2%). ADU = arbitrary densitometric units. Other abbreviations as defined in Figure 1.