Azure c400

The protein expression levels of COX-1, COX-2, and β-actin were determined by Western blot analysis. The gastric epithelial cells were incubated 10 mM acetylsalicylic acid (aspirin, Sigma-Aldrich) for 4 h and then treated with each fraction of A. indica (0.5 μg/mL) for 24 h. Cell lysates were lysed with 100 μL RIPA and resolved by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for Western blot analysis. After blocking by 5% of skim milk at room temperature, the membranes were incubated with primary antibodies against COX-1, COX-2, and β-actin, respectively. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Millipore). The proteins of interest were identified using ECL Western blotting analysis reagent (BIOMAN, Taipei, Taiwan) and analyzed by Azure C400 (Azure Biosystems, Dublin, CA, USA).

A549 cells were infected with DENV-2 luciferase virus at MOI of 5 and incubated for 72 hours. The cells were then harvested by centrifugation and washed twice with chilled phosphate-buffered saline (PBS). The collected cells were resuspended in PBS containing 0.1% NP-40, homogenized using sonication, and centrifuged to separate the soluble fraction. The protein concentrations were determined using the bicinchoninic acid (BCA) assay and diluted to a concentration of 2 mg/mL in PBS.
The proteome samples were mixed with FP and incubated at 25°C for 1 hour. Click reactions were performed using 200 µM TAMRA-azide or biotin-azide, 1 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 100 µM Tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (TBTA), and 1 mM CuSO₄ at 25°C for 30 minutes. The samples labeled with FP and TAMRA-azide were analyzed using SDS-PAGE and imaged on an Azure Biosystems instrument (Azure C400, USA). The samples labeled with biotin-azide samples were incubated with streptavidin agarose resins (Thermo Fisher Scientific, USA) for 3 hours, followed by centrifugation and washing with 0.2% SDS in PBS (5 mL for 10 minutes), PBS (3 times with 5 mL each), and water (3 times with 5 mL each). The samples were then boiled, fractionated using SDS-PAGE, and silver stained.

BALF prepared from each mouse in the same group were pooled into one sample and analyzed by Proteome Profiler Array (R&D Systems). Images were captured using an Azure C400 (Azure Biosystems, CA). The quantifications of each dot were measured by Image J, and the fold changes were calculated by Log₂. The expression levels of cytokines were expressed as the average signal intensity of duplicate spots subtracted from signal background and normalized to total protein concentration.