Fig. 3. Time course of phosphorylation of JNKs and c-Jun in samples from isolated perfused Rana ridibunda hearts. (A) Upper panel: phospho-JNKs were detected in extracts (50 µg of protein) from control hearts (0.5 h equilibration at 25°C followed by 0.5 h perfusion at 25°C) or hearts perfused after equilibration at 42°C for increasing periods of time as indicated. (C) Upper panel: phospho-c-Jun was detected in corresponding samples. The membranes were re-probed with an anti-actin antibody so as to verify equal protein loading (A,C: bottom panels). Densitometric analysis of phospho-JNKs (B) and phospho-c-Jun (D) bands was performed by laser scanning. Western blots shown are representative of at least three independent experiments while data are means ± s.e.m. for at least three independent experiments. ^**P<0.01 and P<0.001 vs control (untreated) hearts.