Fig. 3.

Phosphorylation and nuclear translocation of ERK1/2 and p53 following doxorubicin treatment in H9c2 cells. Cells were maintained in medium with or without 1 μM doxorubicin for 24 h. p-ERK1/2 (A) and p-p53 (B) proteins were measured by indirect immunofluorescence using fluorescein-conjugated secondary antibodies (p-ERK1/2, green; and p-p53, red). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Experiments were carried out at least 3 times, and the representative pictures are shown. The merge images demonstrate nuclear translocation of p-ERK1/2 and p-p53 after phosphorylation by doxorubicin. Cytosolic and nuclear proteins were processed for Western blot analysis to detect p-ERK1/2 and p-p53. Representative Western blots are shown at top right. The optical density is expressed in arbitrary units normalized against a control nuclear sample. Data in histograms represent means ± SE from 6 experiments. *P < 0.05, compared with cytosol of the same treatment group; †P < 0.05, compared with the same cell compartment of the untreated control.