Ab169540

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.

Protein extracts were obtained by lysing cell pellets at 100 °C for 10 min in 2× Laemmli buffer (125 mM HCl-Tris, pH 6.8, 4% SDS, 0.02% bromophenol blue, 20% glycerol, 200 mM DTT). Cellular extracts were then sonicated in a Bioruptor (Diagenode, Liège, Belgium) for 1 min at high intensity. Protein extracts were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham^TM Protran^® 0.45 µm, GE Healthcare Chicago, IL, USA). The membranes were blocked for 1 h with 5% skim milk in TTBS (15 mM HCl-Tris, pH 7.5, 200 mM NaCl, 0.2 M NaCl, 0.1% (v/v) Tween-20), followed by incubation with primary anti-NDUFS1 (Abcam, Cambridge, UK, ab169540, 1:10,000 dilution), anti-NDUFS2 (Abcam, ab103024, 1:2000 dilution), anti-NDUFV2 (Abcam, ab183715, 1:2000 dilution) and anti-GAPDH (Santa Cruz, sc-47724, 1:1000 dilution) at 4 °C overnight.
After washing with TTBS buffer, the membranes were incubated with HRP-conjugated secondary antibody (Bio-Rad, Hercules, CA, USA) for 1 h at a 1:5000 dilution at room temperature. Proteins were detected using an enhanced chemiluminescence detection kit (Pierce^TM Super-Signal West Pico, Thermo Fisher Scientific, Waltham, MA, USA) in a ChemiDoc^TM Touch Imaging System (Bio-Rad) and the relative intensity value quantified with the Image Lab software provided with this system.

The following specific antibodies were used for Western blot analysis: anti-UQCRFS1 (1:1000 dilution, ab14746, Abcam), anti-NDUFS1 (1:1000, ab169540, Abcam), anti-VDAC1 (1:3000, ab14734, Abcam), OXPHOS cocktail (1:1000, ab110413, Abcam), anti-LC3 (1:1000, ab51520, Abcam), anti-PINK1 (1:500, ab23707, Abcam), anti-parkin (1:500, #4211, Cell Signaling Technology), anti-ubiquitin (1:1000, P4D1, BioLegend), anti-PGC-1α (1:1000, ab106814, Abcam), anti-PPARα (1:1000, GTX101098, GeneTex), anti-TFAM (1:1000, GTX103231, GeneTex), anti-ATP5A (1:1000, ab14748, Abcam), anti-SDHA (1:1000, GTX101689, GeneTex), anti-SDHB (1:2000, GTX104628, GeneTex), anti-SDHC (1:500, ab155999, Abcam), and anti-SDHD (1:500, ab189945, Abcam) antibodies.