Transforming growth factor β1

Frozen liver tissues were homogenized in buffer containing 0.25-M sucrose and 10-mM phosphate (pH 7.4). Nuclear fractions were extracted from parts of frozen liver samples using CelLytic^TM NuCLEAR^TM Extraction Kits (Sigma-Aldrich Japan, Tokyo, Japan). Samples were then subjected to 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were transferred to polyvinylidene difluoride membranes as described previously [23 (link)]. Membranes were then incubated overnight at 4°C with primary polyclonal antibodies against alpha smooth muscle actin (α-SMA), cytochrome (CYP)7A1, CYP27A1 (Abcam plc, Cambridge, UK), constitutive androstane receptor (CAR; GeneTex, Inc., Irvine, CA), bile salt export pump (BSEP), CYP7B1, CYP8B1, farnesoid X receptor (FXR), pregnane X receptor (PXR), small heterodimer partner (SHP), sulfotransferase (SULT)2A1, transforming growth factor (TGF)-β1 (Santa Cruz Biotechnology, Santa Cruz, CA), or multidrug resistance-associated protein 3 (MRP3; Sigma-Aldrich Japan). GAPDH (Santa Cruz Biotechnology) and TATA binding protein (TBP; Abcam plc) were used as loading controls for homogenates and nuclear fractions, respectively. Protein signals were detected using ECL Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).

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.