Clarity western enhanced chemiluminescence substrate

Protein samples were resuspended in the SDS sample buffer, boiled at 95°C for 10 min, separated by 12% Mini-Protean TGX precast protein gels (Bio-Rad), and transferred to nitrocellulose membranes (Bio-Rad) using a Trans-Blot semidry transfer cell (Bio-Rad). Membranes were blocked with 5% milk powder overnight at 4°C and incubated for 1 h with monoclonal Anti-Flag M2-peroxidase antibody (Sigma-Aldrich, 1:1,000 dilution) or His₆ tag monoclonal antibody (Thermo Fisher Scientific, 1:2,000 dilution) at room temperature. Afterward, the membranes were further incubated with anti-mouse IgG(H+L)/HRP conjugate (Promega) for another 1 h. After three washes with TBST, chemiluminescence was detected by using Clarity Western enhanced chemiluminescence substrate (Bio-Rad). Protein samples were visualized by Coomassie staining in parallel to show the equivalent protein loading in each lane. Quantitation of Western blot band intensities was done using the CLIQS software (TotalLab). Each Western blot analysis was performed at least three times.

LG powder was provided by Agrisan Company (Larciano, Pistoia, Italy). The liposome components Soy lecithin and Eudragit^® L100 were purchased from Evonik Industries AG (Essen, Germany). Streptozotocin (STZ; cat. S0130) was purchased from Sigma Aldrich-Merck (St. Louis, MO, USA). RIPA lysis buffer (cat. sc-24948A) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA), while the phosphatase and proteinase inhibitor cocktails were obtained from Roche Applied Science (Indianapolis, IN, USA). Micro BCA Protein Assay (cat. 23235) and β-mercaptoethanol (cat. J66742) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). SDS (cat. A02263) and non-fat milk (cat. A0830) powders were obtained from PanReac AppliChem (Milano, Italy). Clarity western enhanced chemiluminescence substrate (cat. 1705061) was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The specifics of the primary and secondary antibodies used are reported in Table 1.

To determine piglets’ immunoreactivity against an unfermented and fermented soybean meal, Western blots were performed. Total soluble protein of 50 μg each was loaded into an 11% SDS-PAGE as described above and then transferred to an Immobolin-P (Millipore, Billerica, USA) polyvinyl difluoride transfer membrane using the wet transfer method at 100 V for 100 min. The membrane was first blocked with buffer comprised of 5% skim milk, 0.1% Tween 20, and 1× TBS for 1 h before the primary antibody incubation. After several washes with washing buffer (0.1% Tween and TBS), the membrane was incubated with the primary antibody at 4 °C overnight. The primary antibody was prepared against soybean antigenic proteins glycinin and β-conglycinin following a published protocol [41 (link)]. Briefly, plasma was collected from 8-week-old piglets that were previously exposed to feed containing soybean meal for 21 d and developed an immune response to soybean allergens. The primary antibody was comprised of 20 μL of pooled pig serum and diluted to 1:500 with the blocking buffer. Rabbit anti-pig horseradish peroxidase-linked IgE was used as the secondary antibody (1: 100,000; Abcam). Proteins were detected by using Clarity Western Enhanced Chemiluminescence substrate (Bio-Rad Laboratories, USA) according to manufacturer’s instructions.

Whole-cell lysates were prepared, and the total protein content of each measured using a BCA kit (Thermo Fisher Scientific). Protein aliquots (10 μg) were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels. After transferring the proteins to Immobilion-P membranes (Merck Millipore Corporation, Darmstadt, Germany), membranes were blocked with 5% skim milk in Tris-buffered saline with Tween-20 and allowed to react with an anti-COX-2 (1:400; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (1:10,000; Wako, Tokyo, Japan) monoclonal antibodies at 4 °C overnight. Membranes were then incubated with the appropriate horseradish-peroxidase-conjugated secondary antibody (1:20,000; Sigma-Aldrich, St. Louis, MO, USA), followed by detection with the Clarity western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Hercules, CA, USA). Western blot signals were captured using an ImageQuant LAS-4000 mini fluorescence imager (GE Healthcare, Pittsburgh, PA, USA) and quantified using Multi Gauge image analysis software (Fujifilm Corporation, Tokyo, Japan).

Whole proteins were extracted from the brain tissues by the total protein extraction kit (Beijing Solarbio Science & Technology Limited Company, China), and the protein concentration was determined by the BCA protein quantitative kit (Hangzhou Fu De Biological Technology Limited Company, China). After adjusting to the same concentration, the protein samples were added to the corresponding proportion of SDS gel loading buffer, boiled and denatured for 5 min, underwent SDS-PAGE electrophoresis, transferred to 5% skim milk PBST buffer at room temperature for 1 h, and washed in PBST 3 times. Then, the appropriate primary antibody (Table 1) was added and incubated overnight at 4°C. The membrane was washed 3 times with PBST, and a horseradish peroxidase-labeled secondary antibody (Table 2) was incubated for 1.5 h at room temperature and then washed 3 times. Proteins were visualized using a Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a Tanon 5200 Full automatic chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd., Shanghai, China). In the western blot images, one lane represented a pool obtained from several animals. But before this, we have done the western blot of a single animal, and the relative density was measured by that.

Western blot analyses were performed according to previously reported procedures^{6 ,9 (link)}. Briefly, protein extracts from cells treated with PTX- or CDDP- PLGA-CSNP-(RGD+/−) formulations and from control groups were run in sodium dodecyl sulfate-polyacrylamide gels and transferred onto Immobilon PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then blocked using 5% milk in TBST buffer (pH 7.5), followed by blocking with primary antibodies (Cell Signaling Technology) in 1:1000 dilution in 5% milk in TBST buffer (pH 7.5) overnight at 4 °C. The next day, the membranes were washed and blocked with secondary antibodies (rabbit or mouse, depending on the primary antibodies; Sigma-Aldrich, USA) and imaged using Syngene GBOX after incubation with Clarity Western enhanced chemi-luminescence substrate (Biorad, USA). Beta actin was used as the internal control for all western blot experiments. Protein bands from western images were quantified using GelQuant.NET software.

Retinas were separated from enucleated eyes and stored at −80 °C. Six samples for each group, each containing two retinas from two different mice, were used. Retinas were homogenized in RIPA buffer containing phosphatase and proteinase inhibitor cocktails (Roche Applied Science, Indianapolis, IN, USA) and protein concentration was measured with the Micro BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). Samples (30 µg proteins each) were run on 4%–20% SDS-PAGE gels and proteins were then transferred on polyvinylidene difluoride membranes. Blots were blocked for 1 h with 5% skim milk and incubated overnight at 4 °C with primary antibodies listed in Table S1 using β-actin as the loading control. Blots were then incubated for 1 h with HRP-conjugated secondary antibodies (1:5000) and developed with the Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Images were then acquired (ChemiDoc XRS+; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The optical density (OD) of the bands was evaluated (Image Lab 3.0 software; Bio-Rad Laboratories) and data were normalized to the corresponding OD of β-actin or NF-κB as appropriate. All experiments were performed in duplicate.