Computational protocol: A novel coumarin, (+)-3′-angeloxyloxy-4′-keto-3′,4′-dihydroseselin, isolated from Bupleurummalconense (Chaihu) inhibited NF-κB activity

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Protocol publication

[…] After RAW-Blue cells were treated with LPS (1 μg/mL) and various concentrations of Pd-Ib (5, 10, 20 μg/mL) for 24 h, total proteins were extracted by lysis buffer [150 mM NaCl, 1 mM EDTA, 1 % NP-40, 2 mM EGTA, 50 mM Tris (pH 7.4)] with phosphatase and protease inhibitors (Roche, Mannheim, Germany). Cell extracts were centrifuged by centrifugation (5424R, Eppendorf, Hamburg, Germany) at 17,000×g for 15 min at 4 °C. The amount of total protein concentration was quantified by the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, 10–25 μg proteins were loaded and separated on sodium dodecylsulphate-polyacrylamide gel (10 % SDS-PAGE), which were further transferred onto polyvinylidene difluoride membranes. The membranes were incubated with various primary antibodies, namely, anti-COX-2, anti-iNOS, anti-IκB-α, and anti-β-actin (1:5000) in 5 % skim milk (wt/vol) in TBST overnight at 4 °C, after blocking with 5 % skim milk (wt/vol) in Tris-buffered saline-Tween [TBST; 150 mM NaCl, 50 mM Tris, 0.05 % Tween-20 (pH 7.4)] for 1 h. After washing with TBST for three times (10 min for each time), the membranes were blocked with secondary antibodies (1:2000) in 5 % skim milk (wt/vol) in TBST for 1 h, following with TBST washed (3 × 10 min). The amount of immunoreactive proteins was detected by enhanced chemiluminescence ECL reagent and X-ray film. The protein bands were quantified using the ImageJ Software (version 4.1.7, NIH, Bethesda, MD, USA) by their relative intensity compared with the control. [...] Experiments were performed at least three times as indicated. Data were presented as mean ± SD from three independent experiments. Statistical differences among groups were evaluated by one-way analysis of variance (ANOVA) and Duncan’s multiple range test by IBM SPSS Statistics 20 for Windows (SPSS Inc., Chicago, IL, USA). P values <0.05 were considered statistically significant. The dose-dependence was visually determined. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals Silica Gel