Fig. 4

AC6-N regulates the PKA and Src-mediated STAT3 phosphorylation and cardiomyocyte survival. a The indicated mice were subjected to an acute ISO treatment (10 mg/kg, i.p.). Fifteen minutes after the injection, the heart was removed carefully to prepare total lysates and analyzed using Western blot analyses. The levels of phosphorylated STAT3 (P-Y705-STAT3), STAT3, and Tubulin were detected using the indicated antibody. Tubulin was used as a loading control. The phosphorylation level of STAT3 was normalized to the amount of the total STAT3, while the amount of the total STAT3 was normalized to the loading control (α-tubulin, N = 4 per group). b, c, d Adult cardiomyocytes were treated with isoproterenol (ISO, 10 μM) in the absence or presence of a PKA inhibitor (KT-5720, 1 μM, 30 min pretreatment; b), a Src inhibitor (Src inhibitor 1, 1 μM, 5 min pretreatment; c), or a STAT3 inhibitor (cucurbitacin I, 500 nM, 30 min pretreatment; d) for 24 h. The hoechst-positive and propidium iodine (PI)-negative with uncondensed chromatin cells were defined as surviving cells. The data are expressed as the mean ± SEM (N = 3, b; N = 4, c; N = 3, d). ^* P < 0.05 vs. AC6^+/+ in the indicated condition. ^# P < 0.05 vs. the basal level in each group. ^ξ P < 0.05 vs. the ISO-treated cells in each group. e Adult cardiomyocytes were immunostained for AC6 (green) and Src (red). Scale bar, 10 μm. The rightmost panels show the enlarged, merged images of fields in white rectangles. Scale bar, 10 μm