Chemiluminescence detection system

Cells were cultured in 100-mm culture dishes at a density of 2 × 10⁵ cells/mL for 24 h. The cells were pre-treated with various concentrations of lupinalbin A (5–100 µM), incubated for 2 h, and then incubated with LPS (1 μg/mL) for 18 h at 37 °C in 5% CO₂ atmosphere. The cells were harvested and lysed using NP40 cell lysis buffer (with 1 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor cocktail) for 30 min on ice. The protein concentration of the cell lysate was measured using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Aliquots of 30-μg protein were loaded and electrophoresed on 10% SDS-PAGE and then transferred onto nitrocellulose membranes (Hybond ECL Nitrocellulose; GE Healthcare, Chandler, AZ, USA). The membranes were washed once with wash buffer comprising PBS with 0.05% Tween 20 and blocked with blocking buffer comprising PBS with 5% skim milk and 0.05% Tween 20 for 1 h. After blocking, the membranes were incubated with target antibodies (against iNOS, STAT1, and β-tubulin) at 4 °C overnight. After incubation, the membranes were washed and incubated for 2 h at room temperature with HRP-conjugated secondary antibody diluted to 1:2000 in blocking buffer. The proteins bands were detected using a chemiluminescence detection system (GE Healthcare, Bucks, UK).

Human primary myoblasts (CHQ cells) were incubated with medium alone or with 10 ng/ml HGF in pre-coated plates with BSA, FN or LN-111 and after 15 min post-stimulation, extracts were prepared by lysing cells in alkaline lysis buffer (Tris-HCl 40 mM, SDS 1%). Proteins obtained by this procedure were quantified with the BCA protein kit (Pierce Co., UK). Samples (40 μg) were resolved on 10% polyacrylamide gels, and the proteins were electrotransferred to a nitrocellulose membrane (Hybond, Amersham, Buckinghamshire, UK). The membrane was treated with 5% non-fat milk, 0.1% Tween 20 in PBS for 2 h, and then incubated overnight with the anti-HGF antibody diluted in blocking solution. Membranes were washed three times with PBS and incubated with an alkaline phosphatase-coupled secondary antibody. Bands were visualized using BCIP/NBT solution kit and scanned images were analyzed using the ImageJ software (NIH, Bethesda, USA). For detecting signaling pathway of ERK, we used anti-human ERK and phosphorylated-ERK rabbit monoclonal antibody, and antibody binding was revealed using a peroxidase-coupled goat anti-rabbit Ig serum, and the signal was analyzed using a chemiluminescence detection system (GE-Healthcare). Expression levels were normalized against the α-actin signal.