Figure 4

Effect of catecholamine-induced hypertrophy on expression and activation of STAT3 in mitochondria and nucleus. H9c2 cells were maintained in 1% serum media for 18 h. Cells were then incubated with PE (50 µM) or ISO (10 µM) for 48 h. (A) Cytosolic (Cyt), nuclear (Nuc), and mitochondrial (Mit) fractions were analyzed by immunoblotting using antibodies specific for STAT3, pS727-STAT3, pY705-STAT3, STAT1, anti-complex V, anti-α-tubulin. Subcellular localization of STAT3 and phosphorylation of STAT3 were quantified by densitometry. The protein levels were quantified by densitometry and the sum of Cyt, Nuc, and Mit fractions set arbitrarily to 100%. Quantification represents the relative intensity to the control cells; mean ± S.E., n = 3. ^*, P < 0.05 (a-d). (B) H9c2 cells were plated onto coverslips and maintained in 1% serum media for 18 h. Cells were then incubated with PE (50 µM) or ISO (10 µM) for 48 h. After fixation and permeabilization, cells were stained with anti-pY705-STAT3 antibody followed by TRITC-conjugated antibody, respectively as described under "Methods". Coverslips were mounted on a slide and analyzed by fluorescence microscopy. Results are representative images of cells from three independent experiments. Red: pY705-STAT3; Blue: Nucleus (DAPI); Merge: pY705-STAT3 + DAPI.