Westar ecl substrates

The dissected L4 and L6 DRG were rinsed with phosphate-buffered saline (PBS) and lysed in Triton lysis buffer. Being denatured proteins were separated on sodium dodecyl sulphate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membrane on ice at 200 mA for 2 hr. The membranes were blocked with 5% skim milk, 0.1% Tween 20 in tris buffered saline for 30 min at room temperature. Then, the membranes were incubated overnight with primary antibodies at 4°C. Protein (20 μg) was used for Western blot analysis using anti-Wnt3a rabbit polyclonal antibody (1:1,000, GeneTex Inc., Irvine, CA, USA), anti-β-catenin mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-growth associated protein 43 (GAP-43) mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-NF-κB mouse monoclonal antibody (1:1,000, Santa Cruz Biotechnology), anti-TNF-alpha rabbit polyclonal antibody (1:1,000, Sino Biological, Wayne, PA, USA), anti-BDNF mouse monoclonal antibodies (1:1,000, Santa Cruz Biotechnology). For the secondary antibody, horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (1:1,000, GeneTex Inc.) were used. The blotting proteins were detected by using Westar ECL substrates (Cyanagen, Bologna, Italy). Detected band intensity was analyzed using Chemidoc (Bio-Rad, Hercules, CA, USA).

ADSC before osteogenic induction (day 0) and ADSC after osteogenic media treatment (day 3 and day 6) were lysed to obtain cell lysates. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL, USA). Protein samples were denatured, electrophoresed by SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5 % skim milk or 5 % BSA, the membranes were incubated with primary antibodies for c-Met, P-Met, FGF2, Runx-2, osterix, ALP, and GAPDH, followed by anti-immunoglobulin G horseradish peroxidase-conjugated secondary antibody. Blots were developed and visualized using WESTAR ECL Substrates (Cyanagen, Bologna, Italy) and analyzed by chemiluminescence using Fusion Solo X (Vilber Lourmat, Marne-La-Vallée, France). The expression levels were quantified using ImageJ software.