Powerpac universal power supply

Western blot analysis was carried out on extracted tissue samples to verify the downregulation of nNOS expression at the protein level. Twenty microliters of each sample (1 μg/ul) was loaded into a precast Bio-Rad Criterion TGX 4–15% gel in a × 1 Bio-Rad Tris/Glycine/SDS buffer and run at a constant 200 V for 45 min using a Bio-Rad PowerPac Universal power supply. Protein was transferred from the precast Criterion TGX 4–15% gel onto a PVDF membrane using a Bio-Rad TransBlot Turbo transfer system. Membranes were blocked for 1 h at room temperature using a 5% solution of Bio-Rad Blotting-Grade Blocker (#1706404) in × 1 phosphate-buffered saline solution (PBS). Membranes were first incubated with primary antibody overnight at 4 °C using a 1:1000 dilution of rabbit polyclonal anti-nNOS antibody (sc-648; lot D0915; Santa Cruz Biotechnology, Dallas, TX) in 5% Blotting-Grade Blocker solution, followed by incubation with secondary antibody for 1 h at room temperature using a 1:5000 dilution of Cell Signaling Technology® anti-rabbit HRP-linked antibody (7074S) in 5% Blotting-Grade Blocker solution. Membranes were developed using a GE Healthcare ECL Prime Western Blotting Detection Reagent system (RPN2232). Membranes were visualized using a Bio-Rad ChemiDoc MP imaging system and data was quantified using NIH ImageJ software [13 (link)].

Dendrimer-miRNA complexes were formed mixing the maximum non-toxic concentration of the different cationic dendrimers with 100 nM of the desired miRNAs in nuclease-free water (Promega, Madrid, Spain). The formation and stability of dendriplexes were evaluated by agarose gel electrophoresis after 2, 24 and 48 h of incubation at room temperature (RT). Samples were treated with Blue/Orange Loading Dye 6X (Promega, Madrid, Spain) and loaded on a gel with 2% agarose (Sigma, St Louis, MO, USA) and 0.01% GelRed® (Biotium, Fremont, CA, USA) in Tris–acetate-EDTA (TAE) buffer (PanReac AppliChem, Darmstadt, Germany) at 120 mV for 40 min on a PowerPac Universal power supply (Bio-Rad Laboratories, Hercules, CA, USA).

For the purification of gp60, CW proteins were extracted as described above. Approximately 6 mg of proteins was resuspended in 18 mL of hydration buffer at a concentration of 7% (v/v) ampholytes with a pH gradient of 3–10. The suspension of proteins was loaded onto a Rotofor preparative IEF cell (Bio-Rad) and ran for 4 h at a constant power of 12 watts (W) using a Powerpac Universal Power Supply (Bio-Rad). Subsequently, fractions enriched in gp60 were pooled and mixed with hydration buffer to reach 18 mL without further addition of ampholytes. This sample was run on the same equipment for 2.5 h at a constant power of 12 W. Fractions enriched in gp60 were precipitated with 70% (v/v) ethanol at −20°C for 24 h and centrifuged and the resulting pellets were resuspended in 2x buffer [0.125 M Tris-HCl, pH 6.8; 4% (w/v) SDS, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, and 0.002% (w/v) bromophenol blue]. The samples were separated by continuous elution electrophoresis for 6 h at 1 W in a Mini-Prep Cell (Bio-Rad). Fractions enriched in gp60 were pooled and kept in elution buffer [0.3% (w/v) Tris-HCl, 1.4% (w/v) glycine, and 1% (w/v) SDS]. The glycoprotein was monitored along the steps of purification by SDS-PAGE electrophoresis and Western blot using anti-gp60 as primary antibody.

High mobility group protein B1 (HMGB1) is a nuclear factor released from dying cells, and, by activating the pattern recognition receptors (PRR), acts as an “alarmin” that initiates inflammation in response to tissue damage. Media collected from MLEC and HPAEC cells, stressed as described above, was used to detect the amount of HMGB1 released using Western Blot analysis (anti-HMGB1 antibody, ab18256 (Abcam, Burlingame, CA) were used in dilution 1:1000, as published)[15 ]. Briefly, media samples were incubated with 6X Laemmli sample buffer (Boston Bioproducts, Ashland, MA), for 5 min at 95°C, loaded on the 4–20% Mini-PROTEAN TGX Stain-Free gels (Bio-Rad Laboratories, Hercules, CA), and electrophoretically separated and transferred using PowerPac Universal power supply and Trans-Blot Turbo transferring system (Bio-Rad Laboratories). The signal was recorded with the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA) using a chemiluminescent protocol and analyzed using Image Laboratory software. The protein loading was normalized per total sample protein using free stain gels, as previously described [23 (link)].