Ab131607

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.

Mouse testis tissues were collected at 24 h after radiation and lysed with RIPA lysis buffer. The protein concentrations were measured by the BCA protein detection kit. Western blotting was performed according to standard procedures. Briefly, equal amounts of the protein were separated by SDS‐PAGE, followed by transfer to polyvinylidene fluoride membranes (Bio‐Rad, USA). The membranes were incubated with antibodies against the proteins Bax (1:800, WL01637; Wanleibio, CHN), Bcl‐2 (1:500, WL01556; Wanleibio, CHN), β‐tublin (1:1000, 10094‐1‐AP; Proteintech), Tfam (1:2000, ab131607; Abcam, UK), PGC‐1α (1:1000, ab54481; Abcam, UK), and Nrf1(A5547; Abclonal, CHN) overnight at 4°C (all from Proteintech, USA). After being washed three times with TBST, the membranes were incubated with secondary antibody (HRP‐conjugated goat anti‐rabbit IgG, SA00001‐2, 1:5000; Proteintech, USA) for 1 h at room temperature. After final washes, the gray values of target bands were analyzed using Image J (version1.51; National Institutes of Health, USA) with β‐tublin as an internal control.

Western blot assay was performed as previously described^{20 (link)}. Tissue or cell extracts were prepared using a dounce homogenizer in cold RIPA buffer supplemented with cocktail. The homogenates were centrifuged at 12,000 rpm for 15 min, and the protein concentrations were determined using a protein assay from Thermo. The protein lysates were separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane. The membrane was scanned using the Image Lab statistical software (Bio-Rad). Antibodies against PAR (Trevigen, 4335-MC-100), PARP1 (Trevigen, 4338-MC-50), p-γH2AX (Ser139) (Trevigen, 4418-APC-020), Gabarapl1 (Abcam, ab86497), ATG12 (Abcam, ab109491), P62 (Cell signaling Technology, 39749), LC3 (Cell Signaling Technology CST, 3868), p-P53 (Ser15) (Cell signaling technology, 9284), p-ATM (Ser1981) (Thermo Fisher MA1-2020),FoxO3a (Cell signaling technology, 12829), pFoxO3a (Cell signaling technology, 9466), β-actin (Cell signaling technology, 3700), PGC1α (Abcam, ab54481), TFAM (Abcam, ab131607), NRF1 (Abcam, ab175932), Lamin B1 (Cell signaling technology,13435), α-tubulin (Cell signaling technology,3873) were used as primary antibodies.

Total protein was extracted with cell lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), and the concentration was measured with BCA kit (Beyotime). The proteins were separated with 12% SDS-PAGE and transferred to PVDF membranes (ThermoScientific). The membranes were separately incubated with primary antibodies overnight at 4 °C, and anti-rabbit (A7016) or anti-mouse (A0216) HRP-conjugated secondary antibodies (Beyotime) for 1 h at room temperature^{49 (link)}. Subsequently, the membranes were washed and the signals were visualized with Clarity ECL Substrate (Bio-Rad, Hercules, CA, USA). Gray-scale results were normalized against β-actin for semiquantitative analysis. The antibodies against IRF1 (sc-514544x), PPARα (sc-130640), and β-actin (sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against PGC1α (ab54481), Pit1 (ab10545), Pit2 (ab191182), phosphorylated FGFR1 (p-FGFR1, ab59194), total FGFR1 (t-FGFR1, ab824), phosphorylated FGFR4 (p-FGFR4, ab192589), total FGFR4 (t-FGFR4, ab41948), NRF1 (ab175932), and TFAM (ab131607) were obtained from Abcam Biotechnology (Cambridge, MA, USA). All of the antibodies were diluted 1:1000 for western blot. Uncropped and unprocessed scans of all blots are provided in Supplementary Figs. 17–21 in the Supplementary Information.

Protein extraction and western blot analysis were conducted as described before.³¹ The primary antibodies included: anti‐TFAM antibody (ab131607, 1:1000), anti‐ATPB antibody (ab14730, 1:5000), anti‐GPX4 antibody (ab125066, 1:3000) and anti‐HMGB1 antibody (ab79823, 1:10000) from Abcam (Cambridge, UK), anti‐BCL2 antibody (3498, 1:1000), anti‐LC3B antibody (3868, 1:1000) and anti‐P62 antibody (5114, 1:1000) from Cell Signaling Technology (Danvers, MA, USA), HRP‐conjugated mouse anti‐ACTB (KC‐5A08, 1:5000), and HRP‐conjugated mouse anti‐GAPDH (KC‐5G5, 1:5000) from Aksomics (Shanghai, China).
The HRP‐conjugated secondary antibodies involved anti‐mouse IgG (KC‐MM‐035, 1:5000) and anti‐rabbit IgG (KC‐RB‐035, 1:5000) from Aksomics (Shanghai, China). The protein bands were visualized with the ChemiDoc MP system (Bio‐Rad, CA). ImageJ software was applied to quantify the band intensity, and the results are presented as the ratio of the target protein to GAPDH or ATCB.

Equal amount of Gastrocnemius muscle tissue (~ 10 mg) was homogenized in 1 mL of TRIzol reagent and total protein was extracted according to the manufacturer’s protocol. Running SDS-PAGE 10% was used for PPARGC1α and 15% was used to detect TFAM and GLUT4. The proteins were then transferred to PVDF (Bio-Rad, USA) membrane. The membranes were then blocked by 10% BSA in TBST solution with 0.01% Tween (v/v) for TFAM and GLUT4 and 10% (v/v) BSA + 5% (w/v) skim milk in TBST solution with 0.01% Tween for PPARGC1α at 4 °C overnight. The membranes were incubated with primary antibodies for 1.5 h at room temperature. Primary antibodies were used with the following concentrations: 1:1000 for TFAM (ab131607, Abcam, UK), 1:200 for GLUT4 (ab176245, Abcam, UK), 1:1500 for PPARGC1α (ab191838, Abcam, UK), and 1:2500 for β-Actin (A2228, Sigma, USA). The membranes were washed and incubated with appropriate secondary antibodies, Goat Anti-Rabbit IgG H&L (HRP) (1:20,000, Santa Cruz, USA) and HRP-conjugated goat anti-mouse IgG (1:5000, Dako, Japan P0447), for 1 h at room temperature. Finally, the protein bonds were visualized by an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA) and the intensity of the bonds were quantitated by image J software (http://rsb.info.nih.gov/ij/).

Immunohistochemical staining was performed on the paraffin sections. After dehydration, antigen retrieval, quenching of endogenous peroxides, and blocking, sections were incubated overnight at 4°C with polyclonal rabbit anti-WDPCP (HPA, 044144) at 1:50, monoclonal mouse anti-NRF1 at 1:100 (Abcam, ab175932), monoclonal mouse anti-NRF2 at 1:100 (Abcam, ab62352), monoclonal mouse anti-TFAM at 1:50 (Abcam, ab131607), and polyclonal rabbit anti-COX4 at 1:200 (Cell Signaling, 4850S). DAB and hematoxylin were used for staining.
Immunofluorescence staining was performed for ALI membranes and HSECs. In brief, HSECs were incubated overnight at 4°C with monoclonal mouse anti-beta tubulin IV at 1:100 (Abcam, ab11315) and subsequently with Alexa Fluor 488 secondary antibodies (Invitrogen) at 1:200 at room temperature for 1 h, and 40,6-diamidino-2-phenylindole (DAPI) (10 mg/ml, Sigma-Aldrich) for 10 min.
Mitochondrial structure was detected via fluorescent MitoTracker (Invitrogen), which was cultured with HSECs at 1:500 dilution at 37°C for 1 h. Then, the cells were fixed and stained with DAPI for 10 min. HSECs were observed and imaged with a Zeiss LSM 780 confocal microscope.