Ab110413

Proteins from isolated brain microvessels and brain tissue were extracted using RIPA buffer with protease inhibitor and phosphatase inhibitor cocktail (Roche Applied Science, Vienna, Austria) described previously [12 (link)]. Protein samples were separated by SDS-PAGE and transferred to the PVDF membrane (Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk for 1 h and then incubated at 4 ℃, overnight with specific primary antibodies against Crif1 (1:1000, sc-134882, Santa Cruz Biotechnology, CA, USA), Notch1 (1:1000, 3608, Cell Signaling, MA, USA), Hes1 (1:500, sc-166410. Santa Cruz Biotechnology, CA, USA), Total OXPHOS complex (1:1000, ab110413, Abcam), adropin (1:500, NBP1-26387, Novus biologicals, CO, USA), β-actin (1:1000, sc-47778, Santa Cruz Biotechnology, CA, USA). After washing three times for 10 min with TBS/T, membranes were incubated with secondary antibodies (1:1000, Sigma-Aldrich, St. Louis, MO, USA) for 2 h at room temperature. Membranes were washed, and signals were observed using an ECL solution (WEST-ZOL). Images were quantified with ImageJ software.

Western blot analysis was performed for 4-HNE, LRP1, mitochondrial OXPHOS complex proteins, NDUFS1, PDGFR-β, and TFAM. Cell lysates were formed using RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) and centrifuged at 16,100× g for 30 min, and the total protein levels were estimated from the supernatant using a BCA kit (23225, Thermofisher). Western blot samples were obtained using XT sample buffer (1610791, Biorad, Hercules, CA, USA) with DTT and boiled at 95 °C for 10 min. The samples (15–20 µg protein, Eastbourne, UK) were resolved in duplicate 4–12% BIS-TRIS gel (3450125, Biorad, Hercules, CA, USA) under reducing conditions and transferred to the PVDF membrane. Probing was performed against 4-HNE (1:2500; HNE11-S, alpha diagnostic international, San Antonio, TX, USA), LRP1 (1:1000; sc-57351, Santa Cruze biotechnology, Dallas, TX, USA), NDUFS1 (1:1000, ab169540, abcam, Cambridge, UK), total OXPHOS antibody cocktail (1:1000; ab110413, abcam), mtTFA (1:1000, ab131607, abcam), and beta-actin (1:5000; 8H10D10, Cell Signaling, Danvers, MA, USA). The signals were detected using IRDy 68RD goat anti-mouse (1: 10,000; 926-68070, Li-Cor, Lincoln, NE, USA) and IRDye 800 CW goat anti-rabbit (1:10,000; 926-32211, Li-Cor). The protein levels were quantified via densitometric analysis using ImageJ software.