Mini protean 3 electrophoresis system

For western blot analysis, 20 μg of total protein was mixed in 2× Laemmli buffer (Sigma-Aldrich) and denatured at 95°C. The samples were then subjected to electrophoresis on an 8% polyacrylamide gel (Thermo Scientific, Waltham, MA, USA) using a Mini Protean 3 electrophoresis system (Bio-Rad Laboratories, Waltham, MA, USA) for 90 minutes. The proteins were transferred electrophoretically to a PVDF membrane (Millipore Corporation, Cheshire, NH, USA) overnight. The membrane was blocked in non-fat milk powder in PBST followed by an overnight incubation with an anti-mouse p110δ PI3K antibody (Santa Cruz Biotechnology, Santa Crus, CA, USA). The membrane was probed with a secondary antibody solution containing anti-goat IgG peroxidase conjugates prior to detection using a SuperSignal West Pico kit (Thermo Scientific) according to the manufacturer’s instructions. Upon reaction, the membrane was exposed, developed, and fixed on a film. The band intensity was measured by the AlphaDigiDoc RT2 gel documentation system software (Alpha Innotech Corporation, CA, USA).

Protein concentrations of the LV tissues were determined by the BCA protein assay kit (Thermo Fisher, Erembodegem, Belgium). Proteins were separated on a 12% SDS-PAGE gel with a mini protean 3 electrophoresis system (Bio-rad Laboratories, Temse, Belgium), transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked for 2 hours with 5% milk in Tris-buffered solution containing 0.1% Tween-20 (TBS-T) followed by overnight incubation at 4 °C with a specific LOX antibody (1/1000, rabbit polyclonal IgG, Abcam, ab31238, Cambridge, United Kingdom). Horseradish peroxidase-conjugated secondary antibodies (DAKO, Belgium) at a dilution of 1/2000 were used. Both primary and secondary antibodies were diluted in 2% milk-TBS-T. LOX was visualized with the enhanced chemiluminescence (ECL) technique using the Pierce ECL Plus western Blotting Substrate (Thermo Fisher, Erembodegem, Belgium). Data were normalized to β-actin protein levels.

Protein samples of interest were run on 4–20% polyacrylamide gels and subjected to electrophoresis. The gels were electrophoresed in Tris–HEPES running buffer (0.1M Tris, 0.1M HEPES, 3 mM SDS, pH 8) using a Mini-PROTEAN 3 Electrophoresis System (Biorad) at 80 V for 15 min and 120 V for the duration of the run. SDS-PAGE separated proteins were transferred to nitrocellulose membrane in transfer buffer (25 mM Tris, 192 mM glycine, 20% v/v MeOH). Transfer was carried out for 90 min at 80 V on ice. The membranes were stained with the reversible stain Ponceau-S solution to check transfer efficiency and destained in distilled water. The membranes ware then blocked for 2 h in 5% BSA in TBS-T (20 mm Tris, pH 7.6, 150 mm NaCl, 0.1% Tween 20) at room temperature on an orbital shaker. The membranes were subsequently incubated with the appropriate dilution of primary antibody overnight. Following incubation, the membranes were washed three times in TBS-T and incubated with the appropriate dilution of secondary antibody for 1.5 h. After three washes in TBS-T for 15 min, the membranes were developed using Supersignal enhanced Chemiluminesence reagents (Thermo Scientific, IL, USA) and exposed to photographic X-ray film.

The total hepatic cell protein levels were assayed using a BCA Protein Assay kit (Beyotime, Shanghai, China). Lysates (50 μg total protein) were mixed with 5× sample buffer, heated to 100°C for 5 min, and separated using 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The resolved proteins were transferred onto a 0.45 μm PVDF membrane using a Mini-Protean 3 electrophoresis system (Bio-Rad, USA). Non-specific binding sites on the membranes were blocked by incubation with a buffer containing 5% (w/v) non-fat milk. The membranes were probed with primary rabbit anti-mouse antibodies against LDLR (1:2,500; Epitomics, Burlingame, USA), LRP (1:2,500; Epitomics, Burlingame, USA), SDC1 (1:1,000; Abcam, Cambridge, USA), SRB1 (1:5,000; Abcam, Cambridge, USA), and β-actin (1:3,000; Santa Cruz Biotechnologies, Santa Cruz, USA). Immunoreactive bands were detected by incubation with horseradish peroxidase-conjugated secondary antibodies (1:3,000; Santa Cruz Biotechnologies, Santa Cruz, USA) and visualized by chemiluminescence. Protein bands were quantified using the Quantity One software (Bio-Rad, Hercules, USA) and normalized to the corresponding β-actin bands.

SDS-PAGE was performed using acrylamide gels with stacking (T 3.94%, C 2.66%) and separating gel (T 15%, C 2.66%) zones, which were prepared with acrylamide:bisacrylamide 40% (37.5:1) solution (Bio-Rad), according to the Laemmli method as described by Fort et al. [22 (link)], using a Mini-Protean^® 3 electrophoresis system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Gels were run at 70 V for 30 min and then at a constant voltage of 120 V for 45–60 min. The approximate molecular weights were estimated using molecular weights markers from 10 to 220 kDa (BenchMark™ Protein Ladder, Invitrogen, Carlsbad, CA, USA). The gels were fixed with 2.5% gluteraldehyde solution, then stained with Coomassie Blue (0.1% PhastGel Blue R solution in 30% ethanol and 10% acetic acid), distained with 30% ethanol and 10% acetic acid, and preserved in 10% acetic acid and 10% glycerol.
Protein extracts were diluted at a ratio of 1:4 in pH 6.8, 10 mM TrisHCl, 1 mM EDTA, 1% sodium dodecyl sulfate (SDS) and 1% β-mercaptoethanol buffer, and subsequently heated at 100 °C for 5 min and centrifuged at 8000 rpm. Immediately before to the electrophoresis performance, these solutions were diluted to 50% with a buffer containing 1.25 mL Tris HCl 0.05 M pH 8.8, 1% SDS, 2 mL glycerol, and 1.75 mL alcoholic solution of bromophenol blue (0.01%) as tracking dye.

Protein purity was determined by SDS-PAGE [28 (link)] on a 12% or 15% resolving gel and 4% stacking gel using a Mini-PROTEAN 3 electrophoresis system (BioRad, Hemel Hemstead, UK). Samples were loaded in either reduced or non-reduced form. Gels were run at 200 V, 30 mA per gel, for 50 min. Proteins were visualised with Coomassie Brilliant Blue R250 V followed by destaining with methanol: water: acetic acid (30:60:10). Alternatively, proteins were visualised by silver staining as performed by method of Heukeshoven & Dernick [29 (link)].