Ecl chemiluminescence system

The liver tissue was cut into 1 mm³ sections, and the cells were lysed with RIPA buffer. After centrifugation at 12,000 rpm for 20 min, the supernatant was collected. The protein concentration in the supernatant was determined using the Pierce BCA Protein Assay Kit (23225, Thermo Fisher). Equal amounts of proteins (20 μg/lane) were separated by 10% SDS‒PAGE and then transferred to PVDF membranes. The PVDF membranes were then incubated at 4°C with the following primary antibodies: anti-LC3 (CST, 4599; 1:1000), anti-BECN1 (CST, 3495; 1:1000), anti-FTH1 (CST, 4393; 1:1000), anti-GPX4 (CST, 52455; 1:1000), anti-xCT (CST, 12691; 1.1000), anti-cleaved (c)-caspase3 (CST, 9664, 1:1000) and anti-β-actin (CST, 4970; 1:1000). PVDF membranes were washed with TBST buffer (137 mM NaCl, 2.7 mM KCl, 16.5 mM Tris, pH 7.4, containing 0.1% Tween-20) and incubated with secondary antibodies at room temperature for 2 h. After washing again with TBST, protein bands in the blots were detected using an ECL chemiluminescence system (GE Healthcare, US). β-actin was used as an internal control.

Cells were treated with or without dichloromethane fraction (DF) of TAR extracts at the indicated concentrations and were harvested. The collected cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktail on ice for 40 min, and then centrifuged at 12,000 rpm for 10 min at 4°C. The total protein concentrations in each sample were measured with a BCA protein assay kit. For western blot analysis, the protein lysates were denatured in Laemmli sample buffer. The same amounts of the protein samples were electrophoresed on SDS-PAGE before transfer onto PVDF. After blocked with blocking buffer (5% non-fat milk, 20 mM Tris–HCl, 500 mM NaCl, and 0.1% Tween 20) at room temperature for 1 h, the PVDF membranes were incubated with corresponding primary antibodies at 4°C overnight. After extensive washing with buffer (20 mM Tris–HCl, 500 mM NaCl, and 0.1% Tween 20), the membranes were incubated for 1 h at room temperature with horseradish-peroxidase-conjugated secondary antibodies. The protein-antibody complexes were detected by ECL detection Kit. The signal was visualized using the ECL chemiluminescence system (GE Healthcare). In order to verify the same protein loading, the blots were reprobed with anti-GAPDH antibody in all of the western blot experiments.

SDS-polyacrylamide gel electrophoresis and immunoblot analyses were conducted according to the previously published procedures^{22 (link)}. In brief, colon tissues were centrifuged at 3000g for 3 min and allowed to swell after the addition of lysis buffer in ice for 1 h. The lysates underwent centrifugation at 10,000g for 10 min to isolate the supernatants. Proteins were separated by 6%, 7.5%, or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred onto nitrocellulose membranes (Millipore, Bedford, MA). The membrane was blocked with 5% non-fat dried milk in Tris-buffered saline and Tween 20 (TBST) (20 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1 h, and incubated overnight with primary antibodies at 4 °C. After washing with TBST buffer, membranes were incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody for 1 h at room temperature. The bands were detected utilizing the ECL chemiluminescence system (GE Healthcare, Buckinghamshire, UK). Equal loading of proteins was confirmed by immunoblotting for β-actin. Quantitative analyses were conducted by scanning densitometry of the immunoblots and β-actin normalization.

IgE reactivity to the different allergen samples (N, POL and AM) was tested by an immunodetection assay as previously described [21 (link)]. Briefly, a total protein amount of 30 μg of the different allergen preparations were placed onto nitrocellulose membranes using a Whatmann Univac vacuum manifold system (GE Healthcare, Canton MA, USA) for allergen binding. The membranes were neutralized with a blocking solution (PBS-0.1 % Tween 20; 3 % defatted milk protein preparation) and incubated (16 h at 4 °C) with individual sera diluted 1/10 from grass pollen allergic patients or with a pool of sera at the same dilution. Afterwards, the membranes were washed with PBS-0.1 % Tween 20 and incubated 1 h at room temperature with anti-human IgE monoclonal antibody diluted 1/5000 followed by a 1 h incubation with a rabbit anti-mouse monoclonal antibody conjugated with peroxidase diluted 1/2000 (Dako, Barcelona, Spain). After washing the membranes with PBS-0.1 % Tween 20, the ECL chemiluminescence system was used for reaction development (GE-Healthcare, Canton MA, USA). Volummograms of the reactive spots were analyzed by scanning densitometry using FUJI FILM MultiGauge v3.0 software.

Cell extracts were isolated using RIPA lysis buffer (Roche) and quantitated by BCA protein assay kit (Pierce). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), and then the gel was transferred onto PVDF membranes (Millipore) using a Tank Transfer System (Bio-Rad). The membranes were then incubated with different primary antibodies overnight at 4°C. Species-specific Horseradish Peroxidase (HRP)-conjugated secondary antibodies were used for detecting the primary antibodies. Detection was accomplished using an ECL chemiluminescence system (GE Healthcare) and the images were exposed to films. The anti-synaptophysin (SYP), pAkt, and p4E-BP1 antibodies were obtained from Cell Signaling Technology. The anti-GAPDH antibody was from Santa Cruz Biotechnology.