Anti pink1

Th total protein extracts were prepared from 10 to 15 mg of the deep-frozen heart tissue. To maintain the extract integrity and function, we used Complete Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), Phosphatase Inhibitor Cocktail II (ab201113, Abcam, Waltham, MA, USA), PMSF (1 mM), EGTA (1 mM), and EDTA (1 mM). The proteins were isolated using a 1xRIPA buffer (ab156034, Abcam, Waltham, MA, USA). The concentration of the protein was determined by the Lowry method [15 (link)]. The samples (30–50 µg) were diluted in Laemmli buffer, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Wilmington, USA) [12 (link)]. The relative levels of the detected proteins were visualized using a C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, Nebraska, USA) and normalized to the appropriate loading control. The antibodies used: (ab56788) anti-DRP1 (1:1000), (ab119685) anti-OPA (1:1000), (ab124773) anti-Mitofusin2 (1:1000), (ab54481) anti-PGC1a (1:1000), (cs2132) anti-Parkin (1:1000), (Affinity Biosciences, DF7742) anti-PINK1 (1:250), (Affinity Biosciences, AF5384) anti-SQSTM1/p62 (1:250), (Affinity Biosciences, DF8163) anti-BNIP3L (1:250), and (cs12741) anti-LC3A/B (1:1500). The anti-GAPDH (ab181602) and anti-tubulin (1:2000) (ab4074) antibodies were used as a loading control.

Alda-1 (purity ≥ 98%) was purchased from Good Laboratory Practice Bioscience (Montclair, IL, USA). Superoxide dismutase (SOD) activity and the malondialdehyde (MDA) content were measured using commercial assay kits (Jiangsu Jiancheng Technology Co., Ltd., Nanjing, China). The following antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA): anti-interleukin (IL)-1β (Cat# AF5103), anti-IL-6 (Cat# DF6087), anti-tumor necrosis factor (TNF)-α (Cat# AF7014), anti-vascular endothelial growth factor (VEGF) (Cat# AF5131), anti-ALDH2 (Cat# DF6358), anti-PINK1 (Cat# DF7742), anti-Parkin (Cat# AF0235), anti-Beclin-1(Cat#AF5128), anti-p62(Cat#AF5384), and anti-LC3(Cat#AF4650). Immunohistochemistry kits were purchased from Maixin Biotechnology Development Co. Ltd. (Fuzhou, China). Pathogen-free male Sprague-Dawley rats (n = 36, 200–250 g each, and 2–3 months of age) were supplied by Zhejiang Vital River Laboratory Animal Technology Co. Ltd. (Jiaxing, China).