Proteome profiler kit

The effect of pineapple vinegar on the angiogenic process of 4 T1 cancer cells was investigated using proteome profiler kit (ARY015, R&D System, USA). Briefly, the membranes were blocked with blocking buffer while the protein samples were incubated with the detection antibody at room temperature for an hour. Next, the protein mixtures were transferred to the membrane and incubated at 4 °C overnight. On the next day, the membranes were washed three times using washing buffer and incubated with streptavidin-HRP for 30 min. Lastly, the membranes were washed again 3 times before the substrate was added to develop chemiluminescence. The membranes were then viewed using Chemi Doc XRS (Bio-Rad).

The THP-1 human monocyte cell line was purchased from the American Type Culture Collection. Cells were maintained in culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) at 37°C in a humidified atmosphere of 5% CO₂. The cells were seeded into six-well plates at a concentration of 1 × 10⁶/ml in a whole volume of 4.8 ml/well. The cells were differentiated into macrophages by the addition of 100 ng/ml phorbol 12-myristate 13-acetate (PMA) for 72 h. After differentiation, the cells were washed twice with fresh media w/o PMA and stimulated with ES or whole parasite. For whole parasite stimulation, the cells were maintained in Nunc polystyrene (PS) EasYFlask™ 25 cm² (Thermo Scientific) flasks at the same cell concentration and density per square centimeter. In the case of cells stimulated with parasite antigens and LPS, the cells were first treated with LPS (100 ng/ml), and parasite (one 10-cm worm/10 × 10⁶ cells) or antigens (5 µg/ml) were added after 1 h. After 24 h, the stimulation culture media was collected and cells were washed with sterile PBS. Cells for phosphokinase analysis were lysed with lysis buffer from Proteome Profiler kit (R&D), cells for RNA isolation were directly treated with fenozol supplied with Total RNA kit (A&A Biotechnology) and stored at −80°C until use.

Screening of MAPK activities were performed as follows: SMCs cultured with cytokines for 4 days were lysed and the activities of 24 MAPKs then measured using a ProteomeProfiler kit (R&D Systems). Activation of p38 MAPK and JNK was measured by immunoblot as the ratio of the phosphorylated band to the total band.

We evaluated 35 molecules related to apoptosis in two biological replicates, using two independent supernatants from CLF and two from IPF. Proteins were quantified (Bradford assay) and maintained at −80°C until use. The profile of apoptosis-related proteins was evaluated using the Proteome Profiler™ kit (R&D Systems, Minneapolis, MN, USA). Briefly, 360 μg/protein diluted in 1.5 ml of array buffer (total volume) was added to the membrane and incubated overnight, and posteriorly, a detection antibody cocktail was added. Finally, protein spots were detected and visualized with the Imaging System from Bio-Rad (ChemiDoc™ XRS+ System). Pixel density was analyzed by densitometry using the online ImageJ 1.39c software provided by NIH.