Cyp d

HUVEC were lysed in 50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl (Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), 0.25% sodium deoxycholate (Sigma-Aldrich), protease inhibitors (10 µg/mL Leupeptin, 10 µg/mL Aprotinin and 1 mM Phenylmethyl-Sulfonyl Fluoride, PMSF) (Sigma-Aldrich), and phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium vanadate, 5 mM sodium phosphate) (Sigma-Aldrich). Lysates (40 µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose sheets. Western Blot analysis was performed using antibodies against OPA1, DRP1, LC3 B-I/-II, BECLIN (Cell Signalling, Euroclone, Pero, Italy), CYPD, CPT1A, GLUT1 (Thermo Fisher Scientific), BNIP3 (Sigma-Aldrich), p62 (Invitrogen, Carlsbad, CA, USA), ATGL, PLIN2 and mitochondrial oxidative phosphorylation complexes (OXPHOS) (Abcam, Cambridge, UK). Actin (Santa Cruz, Dallas, Texas, USA) was the control of equal loading. After washing, secondary antibodies labelled with horseradish peroxidase (GE Healthcare, Waukesha, WI, USA) were used. Immunoreactive proteins were detected with Clarity™ Western ECL substrate by ChemiDoc MP Imaging System (Bio-Rad). Densitometry of the bands was performed with ImageJ. The Western blots shown are representative and the densitometric analysis was performed calculating the ratio between the protein of interest and Actin on three independent experiments ± Standard Deviation (SD).

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40 Substitute). Protein concentration was determined using the Bradford reagent (Sigma Aldrich). Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes by using Trans-Blot Turbo^TM Transfer Pack (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed using primary antibodies against Pgp, PON2, TXNIP, CYP D (Thermo Fisher Scientific), SOD2, OPA1 (BD Biosciences, St. Diego, CA, USA), TRPM7 (Bethyl, Montgomery, TX, USA), MagT1 (Abcam, Cambridge, UK), SIRT2 (Merck Millipore, Burlington, MA, USA), DRP1 (Cell Signalling, Danvers, MA, USA), β-actin and GAPDH (Santa-Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies conjugated with horseradish peroxidase (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) were used. The immunoreactive proteins were detected with Clarity^TM Western ECL substrate (Bio-Rad) and images were captured with a ChemiDoc MP Imaging System (Bio-Rad). Densitometry of the bands was performed with the software ImageJ (National Institute of Health, Bethesda, MD, USA). The Western blots shown are representative and the densitometric analysis was performed on three independent experiments ± SD.

HUVEC were lysed in 10 mM Tris-HCl (pH 7.4) containing 3 mM MgCl₂, 10 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.5 mM EDTA and protein inhibitors, separated on SDS-PAGE and transferred to nitrocellulose sheets at 400 mA for 2h at 4 °C. Western blot analysis was performed using antibodies against P-HSP27, HSP27, DRP1, pDRP1^Ser616, pDRP1^Ser637, OPA1 (Cell Signaling Technology, Danvers, MA, USA), TXNIP, CYP D, ZO-1, VECAD (Thermo Fisher Scientific), HSP70 and Actin (Tebu Bio-Santa Cruz, Magenta, Italy), which were used as a control of loading. After extensive washing, secondary antibodies labeled with horseradish peroxidase (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) were used. The Super-Signal chemiluminescence kit (Thermo Fisher Scientific) was used to detect the immunoreactive proteins. All the experiments were performed at least three times, and a representative blot is shown. Densitometry of the bands from three blots was performed with the software ImageLab (Bio-Rad, Hercules, CA, USA), and the results are expressed as the mean ± SD.