Western blotting
The dogs in the three groups were euthanized at the end of treatment.
Their hearts were excised, and the LV was collected. The LV was quickly
frozen in liquid nitrogen and stored at -80℃ until further use. LV
tissue was lysed in lysis buffer [Cell Signaling Technologies (CST),
MA, USA] containing a protease inhibitor and phosphatase inhibitor
cocktail (Thermo Scientific, IL, USA) and was homogenized with beads in
a Bullet Blender (Next Advance, NY, USA). After centrifugation at 13,000
x g for 5 min at 4°C, the supernatant was collected, and protein
concentrations were determined by a Bradford assay (Cat. 500-0113,
Bio-Rad, PA, USA). Equal amounts of protein (90 µL) were mixed with 30
µL of 4X NuPAGE LDS sample buffer (Cat. NP0008, Thermo Fisher, CA, USA)
and 15 µL of 10X NuPAGE reducing agent (Cat.NP0009, ThermoFisher, CA,
USA), boiled, separated on NuPAGENovex 4%–12% Bis-Tris Protein Gels
(Cat. WG1402B, CA, USA), and transferred to nitrocellulose membranes
(LiCor, NE, USA) using the NuPAGE electrophoresis system (ThermoFisher,
CA, USA). Membranes were blocked using Odyssey blocking buffer (LiCor,
NE, USA) for 1 h at room temperature before incubation with primary
antibodies overnight. The membranes were then washed with 1X PBST (0.1%
Tween 20 in Tris-buffered saline) and incubated with secondary antibody
for 1 h at room temperature. The signal was detected using an Odyssey
scanner (LiCor, NE, USA). The primary antibodies used were TGF-β,
p-ERK1/2), ERK1/2, matrix metalloproteinase-9 (MMP-9), angiotensin II
type I receptor (AT-1R), and glyceraldehyde 3-phosphate dehydrogenase
(CST, MA, USA). The secondary antibodies used were goat anti-mouse IRDye
800 (LiCor, NE, USA) and IRDye 680 goat anti-rabbit (Rockland, PA, USA).