Sc 2048

Liver tissues were homogenized in lysis buffer (50 mmol/L Tris‐HCl [pH 7.4], 150 mmol/L NaCl, 5 mmol/L EDTA, 1% (v/v) Nonidet P‐40, 0.1% (w/v) sodium dodecyl sulfate, 0.5% [w/v] sodium deoxycholate) including protease inhibitor cocktail (P3100‐010, GenDEPOT, TX), incubated on ice for 20 min, and centrifuged for 20 min at 13,000 xg at 4°C. Approximately 20 μg of total protein was subjected to 16% SDS‐PAGE and transferred to nitrocellulose blotting membrane. The membranes were blocked with 5% (w/v) skim milk in TBS‐T buffer (140 mmol/L NaCl, 25 mmol/L Tris‐HCl [pH 7.4], 2.7 mmol/L KCl, 0.05% [v/v] Tween 20) and incubated with the antibodies for UQCRH (ab134949, Abcam, Cambridge, UK) and α‐tubulin (sc‐5286, SantaCruz, CA) overnight at 4°C. After washing three times with TBS‐T, the membranes were incubated with a horseradish peroxidase‐conjugated secondary antibody (A120‐101P and A90‐116P, Bethyl Laboratories, TX) at room temperature for 1 h. The membrane was reacted with western blotting luminol reagent (sc‐2048, SantaCruz).

The total cellular extracts of LECs were prepared in ice-cold radioimmune precipitation buffer (RIPA buffer), and Western analysis was carried out as described previously [7 (link),70 (link),71 (link),72 (link)]. The membranes were probed with anti-Nrf2 (sc-722, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Nrf2 (ab62352, Abcam^®, Cambridge, MA, USA), Anti-Klf9 (ab177158, Abcam^®, Cambridge, MA, USA), Anti-Klf9 (sc-12996, Santa Cruz Biotechnology, Dallas, TX, USA), Anti-Prdx6 antibody (LF-PA0011, Ab Frontier, South Korea), Tubulin (ab44928, Abcam^®, Cambridge, MA, USA) or β-actin (A2066, Sigma-Aldrich, St. Louis, MO, USA) or Lamin B1 (ab133741, Abcam^®, Cambridge, MA, USA) as an internal control to monitor levels of protein expressions. After secondary antibody (sc-2354 and sc-2768, Santa Cruz Biotechnology, Dallas, TX, USA) incubation, protein bands were visualized by incubating the membrane with luminol reagent (sc-2048; Santa Cruz Biotechnology, Dallas, TX, USA). Finally, images (bands) were recorded with a FUJIFILM-LAS-4000 luminescent image analyzer (FUJIFILM Medical Systems Inc., Hanover Park, IL, USA).

Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC cell lines (American Type Culture Collection - Manassas, VA – United States) and cultured in growth medium (Dulbecco’s modified eagle’s medium – DMEM, Vitrocell, 00025) supplemented with fetal bovine serum (FBS) 10% from Gibco (12657029, South America) and used at passages 4–6. After serum deprivation in culture medium for 12 h, confluent HUVECs were treated with dextrin (10 mM) for 60 min or not (control) in the presence of BH₄ (100 μM, for 30 min), L-arginine (1 mM, for 30 min), or BH₄ plus L-arginine. Then, the cultured cells were lysed on ice in RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors, and the lysates were placed under non-denaturing conditions. The samples were prepared with 4x Laemmli sample buffer (Biorad, 1610747) plus betamercaptoethanol 10% (Biorad, 1610710) and were loaded on polyacrylamide gel 8%. During electrophoresis and protein transfer to the nitrocellulose membrane, the buffers were placed in an ice-water bath, and the whole apparatus was kept at 4 °C. The eNOS monomer and dimer forms were incubated with antibody against eNOS (1:1000, BD Bioscience, 610296) and α-tubulin or GAPDH were used to normalize the results. The bands were detected by a chemiluminescence substrate (Santa Cruz, SC-2048).