Angiotensinogen

At 16 weeks of age, the animals were sacrificed by an overdose of anesthesia and the heart was excised. Western blot analysis was performed using lysates from the heart tissues. Proteins were separated and transferred to membranes using standard protocols. Then, they were probed with antibodies against prorenin and renin (1:1000, Santa Cruz Biotechnology, Inc.), (P)RR (1:1000, Abcam), angiotensinogen (1:200, Phoenix Pharmaceuticals, Inc.), angiotensin II AT1 receptor (1:200, Enzo Life Science), extracellular signal-related kinases (ERK)1/2 (1:1000, Cell Signaling Technology, Inc.), phosphorylated (p)-ERK1/2 (1:100; Cell Signaling Technology, Inc.), transforming growth factor (TGF)-ß1(1:2000; Santa Cruz Biotechnology, Inc.), p38 mitogen-activated protein kinase (p38MAPK) (1:1000; Cell Signaling Technology, Inc.), p-p38MAPK (1:1000; Cell Signaling Technology, Inc.), heat shock protein (HSP)27 (1:1000; Santa Cruz Biotechnology, Inc.), and p-HSP27 (1:1000; Santa Cruz Biotechnology, Inc.). The blots were visualized by chemiluminescence (ECL Prime, Amersham), and the signals were quantified by densitometry. α-Tubulin (1:1000, Cell Signaling Technology, Inc.) served as the loading control.

At the end of the experiment at 12 weeks of age, the heart was excised. Western blot analysis was carried out using homogenates from the heart tissues. Proteins were separated and transferred to membranes using standard protocols, after which they were probed with antibodies against prorenin and renin (1:100; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), (pro)renin receptors (1:100; Santa Cruz Biotechnology, Inc.), angiotensinogen (1:200; Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA), angiotensin II AT1 receptor (1:200; Enzo Life Sciences, Inc., Farmingdale, NY, USA), extracellular signal-related kinases (ERK)1/2 (1:100; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated (p)-ERK1/2 (1:100; Cell Signaling Technology, Inc.), transforming growth factor (TGF)- β1 (1:200; Santa Cruz Biotechnology, Inc.), p38 mitogen-activated protein kinase (MAPK) (1:100; Cell Signaling Technology, Inc.), p-p38MAPK (1:100; Cell Signaling Technology, Inc.), heat shock protein (HSP)27 (1:100; Santa Cruz Biotechnology, Inc.) and p-HSP27 (1:100; Santa Cruz Biotechnology, Inc.). The blots were visualized by means of chemiluminescence (ECL; GE Healthcare UK Ltd., Amersham Place, Buckinghamshire, England), and the signals were quantified by densitometry. GAPDH (analyzed with an antibody from Cell Signaling Technology, Inc.) served as the loading control.