Mini protean 2 apparatus

After treatments with R97, R60, SG, or TRU (1 mg/mL) for 1 hour or with insulin (100 nM) for 30 min, rat cardiac fibroblasts were washed with ice-cold PBS and lysed with RIPA buffer containing mammalian protease and phosphatase inhibitor mixtures. Protein concentration was measured by Bio-Rad DC Protein Assay (Bio-Rad Laboratories). Proteins were separated on 4–20% SDS-PAGE Mini-PROTEAN TGX Precast Gels using a Mini-PROTEAN II apparatus (Bio-Rad Laboratories) and electrophoretically transferred to the nitrocellulose membrane (Hybond-C; GE Healthcare). To avoid nonspecific binding, membranes were incubated in blocking buffer containing 5% (w/v) albumin in Tris-buffered saline (TBS)/Tween and probed overnight at 4°C with primary antibodies (anti-phospho-IGF-1R, anti-IGF-1R, anti-phospho-PI3K, anti-PI3K, anti-phospho-Akt, anti-Akt, anti-phospho-AMPK, anti-AMPK, or anti-β-actin as internal normalizers). Nitrocellulose membranes were then washed with TBS/Tween and incubated with horseradish peroxidase-labelled secondary antibodies in 5% albumin TBS/Tween at room temperature for 1 hour and successively washed with TBS/Tween. Chemiluminescence detection was performed using Clarity Western ECL Substrate (Bio-Rad Laboratories). Bands were acquired with a CCD imager (ChemiDoc™ MP System, Bio-Rad) and analyzed by using Image Lab analysis software (Bio-Rad).

Samples of cell lysates containing the equivalent of 1.5 × 10⁴ cells (approximately 15 µg protein) were mixed with sample buffer^{38 (link)}, heated to 95 °C for 10 min, and separated by electrophoresis in a Mini Protean II apparatus (BioRad) using hand cast gradient (7–15% acrylamide) or homogenous (10% acrylamide) SDS-polyacrylamide gels^{38 (link)}. Proteins were visualized by colloidal Coomassie Brilliant Blue staining^{39 (link)}.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of LaE was performed on 12 % polyacrylamide gels using a Mini Protean II apparatus (Bio-Rad, California, USA). Wells were loaded with LaE containing 20 μg/mL of protein, resuspended in SDS-sample buffer and boiled for 4 min, and the electrophoresis carried out as described elsewhere [26 (link)]. Proteins were electrophoretically transferred from the gel to nitrocellulose membranes. The nitrocellulose membranes were cut into vertical strips and blocked for 12 h with 0.15 M phosphate-buffered saline, pH 7.2 (PBS) containing 10 % FBS, at 4 °C. Incubation with mouse sera (diluted 1:1000 in PBS containing 0.05 % of Tween 20 and 10 % of FBS) was carried out during 1 h at room temperature with mechanical agitation. After five 1-min washes in PBS, the membranes were incubated during 1 h with appropriately diluted horseradish peroxidase-conjugated anti-mouse IgG1 or IgG2a (Sigma Chemical Co., Saint Louis, MO, USA) in PBS containing 0.05 % Tween 20 and 5 % FBS, at room temperature. The nitrocellulose strips were finally incubated with a mixture of 3’3-diaminobenzidine (Sigma Chemical Co., Saint Louis, MO, USA) and H₂O₂ in PBS. Naïve mouse sera were used as negative controls.

Thoracic aorta and muscle were homogenized in a buffer consisting of 250 mM sucrose, 1 mM EDTA, 0.1 mM phenyl methylsulfonyl fluoride, and 20 mM potassium phosphate buffer (pH 7.6). Large tissue debris and nuclear fragments were removed by two successive low-speed spins (3500 rpm, 5 min; 8000 rpm, 10 min, 4 °C). Quantity of protein was measured by the Bradford method. An equal amount (35 μg) of protein was separated by 10% Sodium Dodecyl Sulfate (SDS)-PAGE. After electrophosis, protein was transferred electrophoretically to nitrocellulose membranes using a Mini-Protean II apparatus (Bio-Rad, Hercules, CA, USA). The membranes were then blocked by 5% bovine serum albumin (BSA) powder in 0.05% Tween 20-Tris-bufferd saline (TBS-T) for 1 h, and subsequently washed and incubated with primary antibodies to VCAM-1, ICAM-1, E-selectin and ET-1 (in aorta) and Insulin receptor substrate-1 (IRS-1) and glucose transporter type 4 (Glut4) (in muscle) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a final dilution of 1:1000 overnight at 4 °C. After washing with TBS-T, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. Signals were detected by a chemiluminescence (ECL) using detection system (Amersham, Buchinghamshire, UK). The bands were analyzed densitometrically by using a Chemi-doc image analyzer (Bio-Rad, Hercules, CA, USA).

The Detroit 551 cells were cultivated in 6-well plates with the density of 3 × 10⁵ cells/well by α-MEM medium for 24 h. Cells were then cultured for another 24 h in medium supplemented with various testing samples. Then, the cells were washed with PBS buffer and then sonicated with cold lysis buffer (200 μL) for 30 min. In western blot analysis, samples (20 ng protein) were loaded into each well and resolved with 12% SDS-PAGE. Then, the PAGE was electrotransferred onto a membrane of polyvinylidene difluoride (PVDF) using the MiniProtean II apparatus (Bio-Rad Laboratories, Carlsbad, CA, USA). The membrane was incubated with an anti-caspase-3 antibody (primary antibody, BioVision, Inc., Milpitas, CA, USA); then the immune complexes were reacted by the ECL reagents (Millipore, Billerica, MA, USA). The resulting membrane image was collected and analyzed using image analysis software (VIpro Platinum, Version 12.9; UVItec, Cambridge, UK) [22 (link)].

The samples of the hippocampus were homogenized in protein extraction reagent containing protease inhibitors, and the concentration of the protein was determined. The proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis using a Mini‐Protean II apparatus (Bio‐Rad, CA, USA). The proteins were electroblotted onto polyvinylidene difluoride membranes, which were blocked. The membranes were incubated with primary antibodies against BDNF (ab108319, 1:1000), interleukin‐1β (IL‐1β, ab9722, 1:1000), ionized calcium binding adapter molecule 1 (Iba1, ab178847, 1:1000) and tumor necrosis factor‐α (TNF‐α, 11948S, 1:1000) overnight at 4°C. The membranes were further incubated with horseradish peroxidase‐conjugated anti‐rabbit IgG (7074S, 1:5000) and developed using ECL reagents. The chemiluminescence signal was imaged by a ChemiDoc XRS system (Bio‐Rad), and the protein band signals were evaluated using ImageJ 1.4.3.67 software. The signals of individual protein bands were normalized to the signal intensity of the β‐actin band and are presented in arbitrary units.

Whole-cell extracts were prepared by lysing 5 × 10⁶ cells in 50 µL 2× Laemmli sample buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue and 0.125 M Tris-Cl, pH 6.8). A 15 µL aliquot of cell lysate was heated at 95 °C for 10 min and then loaded onto 10% stain-free polyacrylamide gels for SDS-PAGE using a Bio-Rad MiniProtean II apparatus. The proteins thus separated were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA) with the help of the Bio-Rad Mini Trans-Blot electrophoretic transfer cell. The membrane was probed with rabbit polyclonal anti-SdhA (1:1000, custom-made), and anti-SdhC (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) diluted in blocking buffer (1% casein, in TBST) overnight at 4 °C, followed by incubation in secondary goat anti-rabbit, HRP-conjugated (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at RT. The protein bands of interest were visualized using Clarity ECL Western blotting substrate (Bio-Rad, Hercules, CA, USA) and acquired on an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA) or a Chemidoc Touch (Bio-Rad, Hercules, CA, USA).