Tropomyosin

Tissue extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, USA). The resulting preparations (10 to 20 μg) were then fractionated by polyacrylamide-SDS gel electrophoresis and immunoblotted with SIRT-1 (sc-15404), PGC-1β (sc-373771), ATP5B (sc-55597), MuRF1 (sc-32920), MAFbx (sc-33782), tropomyosin (sc-28543), MYH (sc-20641), hypoxanthine phosphoribosyltransferase (HPRT) (sc-20975), NOX2 (gp91-phox) (sc-130543), and β-actin (47778) from Santa Cruz Biotechnology, Inc., USA; with UCP-3 (catalog no. 97000), pAMPKβ1 (Ser182) (catalog no. 4186), and AMPKβ1 (catalog no. 4150) from Cell Signaling Technology, USA; with ATG5 (NBP2-24389) and LC3B (NB100-2220) from Novus Biologicals, USA; and with anti-SQSTM1/p62 (catalog no. ab56416) from Abcam, USA. The primary and secondary antibodies were used at dilutions of 1:1,000 and 1:10,000, respectively. The blots were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. The immunoreactive bands were analyzed using ImageJ software, and a densitometry analysis was conducted.

The sections were stained with hematoxylineosin or prepared for immunohistochemical/fluorescence staining. Monoclonal antibodies specific for von Willbrand factor (vWF; Dako), tropomyosin (Santa Cruz Biotechnology), and a-sarcomeric actinin (Sigma) in pigs were used for immunohistochemical staining. The vWF-positive capillaries were counted under 200× microscopy. Twenty fields were randomly selected in each patch. Capillary density was expressed as the mean number of vessels per square millimeter.^{4 (link)}