Quantione 1 d analysis software

The C6 cell lysates (20 µg) were electrophoresed and transferred to the nitrocellulose membrane. Nitrocellulose membranes, blocked in 5% of non-fat milk in PBS 0.1% Tween-20, were probed with mouse monoclonal anti-β actin antibody (antibody dilution 1:5000) (A5316 Sigma, MO, USA), rabbit polyclonal anti-VEGF anti-NOS-2 antibodies (antibodies dilution 1:200) (sc-152, sc-651, respectively, both purchased by Santa Cruz biotechnology, CA, USA), mouse monoclonal anti-HIF-1α, anti-MMP-9, anti-MMP-2, anti-NOS-1 antibodies (antibodies dilution 1:200) (sc-5354, sc-393859, sc-13595, sc-5302, respectively, all purchased by Santa Cruz biotechnology, CA, USA), then incubated in the presence of specific enzyme conjugated IgG horseradish peroxidase. Immunoreactive bands were detected by ECL system (Amersham Int., Buckunghamshire, UK) and analyzed by densitometry. Densitometric values, expressed as Integrated Optical Intensity (IOI), were estimated in a CHEMIDOC XRS system by the QuantiOne 1-D analysis software (BIORAD, Richmond, CA, USA). Values obtained were normalized based on densitometric values of loading control β-actin.

DPSC lysates (20 μg/sample) were electrophoresed on a 4–20% SDS-PAGE gel (ExpressPlus™ 10x8, GenScript Biotech Corporation, China) and transferred to nitrocellulose membranes further blocked in 5% of nonfat milk, 10 mmol/l Tris pH 7.5, 100 mM NaCl, and 0.1% Tween 20. Membranes were then probed for mouse anti-β-actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) (primary antibody dilution 1 : 10,000) and mouse monoclonal anti-BSP II (Santa Cruz Biotechnology, CA, USA) (primary antibody dilution 1 : 200) and incubated in the presence of specific conjugated IgG horseradish peroxidase. Immunoreactive bands were identified using the ECL detection system (Amersham Int., Buckinghamshire, UK) and analyzed by densitometry. Densitometric values, expressed as the percentage of the integrated optical intensity ratio of BSP II and β-actin, were estimated by the ChemiDoc XRS system using the QuantiOne 1-D analysis software (Bio-Rad, Richmond, CA, USA).

The western blowing analysis of Bax and Cyt C was carried out as previously reported [59 (link)]. Specifically, CTX-TNA2 lysates (20 µg) were boiled fof 5 min in sample buffer, made of tris-HCl pH 6.8, glycerol, 2-mercaptoethanol, SDS and bromophenol blue. They were then electrophoresed and transferred to nitrocellulose membranes. Nitrocellulose membranes then were blocked in 5% non-fat milk, 10 mmol/L Tris pH7.5, 100 mmol/L NaCl, 0.1 % Tween 20, probed with mouse monoclonal anti-β-tubulin antibody (cat. number T4026, 1:1000; Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-Bax antibody (cat. number sc-7480, 1:200), mouse monoclonal anti-cytochrome c antibody (cat. number sc-13156, 1:200) (both from Santa Cruz Biotecnologies, Heidelberg, Germany) and incubated in the presence of specific enzyme conjugated IgG horseradish peroxidase. Immunoreactive bands were identified using the ECL detection system (Euroclone, Pero, Milano, Italy) and analyzed by densitometry. Densitometric values were quantified by the CHEMIDOC XRS system and expressed as Integrated Optical Intensity using the QuantiOne 1-D analysis software (Bio-rad Laboratories, Richmond, CA, USA). Values obtained were normalized on densitometric values of internal β-tubulin. Results are expressed as the mean ± SD.