Examination of cell signaling pathways by quantitative western blot

The modulation of signaling pathways by LCM was examined by western blot as previously described with minor modifications [26, 28]. AGS cells (2.0 × 10⁶cells/well) were cultured in a 6-well plate as described above. AGS cells were stimulated with H. pylori ATCC 43504 (6.0 × 10⁸ CFU/well) in the presence or absence of LCM (5 % v/v) for 0.25, 0.5, 1, 2, and 3 h. Proteins were extracted from whole cell lysates of AGS-treated cells using Mammalian Protein Extraction Reagent (M-PER, Pierce Biotechnology, Illinois, USA) supplemented with Halt protease and phosphatase inhibitors (Pierce Biotechnology, Illinois, USA) according to the manufacturer’s instructions. Protein concentrations from cell lysates were determined using the Pierce® BCA protein assay kit (Pierce Biotechnology, Illinois, USA). Cell extracts were fractionated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad, Philadelphia, USA) and blocked with 10 % non-fat milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05 % Tween 20). Blocked membranes were incubated with mouse antibodies against NF-κB (p65), phospho-NF-κB (p65), c-Jun, phospho-c-Jun (Santa Cruz Biotechnology, California, USA) and β-actin (Cell signaling Technology, Inc, MA, USA), washed with TBST and incubated with horseradish peroxidase-labeled goat anti-mouse secondary antibodies for 1 h. Peroxidase signals were measured and imaged by ChemiDoc™ XRS (Bio-Rad, Philadelphia, USA). Densitometric analyses for protein quantification were carried out using ImageJ 1.45 s software.