Ecl select detection system

A549 cells were harvested 24–60 h after cytokine stimulation depending on the parameters of the experiment. After washing with phosphate-buffered saline (PBS), cells were directly lysed in 2 × sodium dodecyl sulfate(SDS) sample buffer without bromophenol blue dye (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% β-mercaptoethanol, 25 mM ethylenediaminetetraacetic acid (EDTA)). Protein concentration was determined using a protein quantification assay kit (Macherey-Nagel, Duren, Germany). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. After blocking with 2% ECL advance blocking reagent™ (GE Healthcare, NJ, USA) in PBS containing 0.1% Tween 20 (polyoxyethelenesorbitan monolaurate), the membranes were incubated overnight with primary antibodies at 4 °C. After washing and incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG, the antigen-antibody complexes were detected using chemiluminescence (ECL select detection system™, GE Healthcare) and then visualized using an Imagequant Las4000 system™ (Bio-Rad, CA, USA).

ECL® Select Detection System was purchased from GE Healthcare (Milan, Italy). Anti-mouse IgG HRP-linked (1:5000) was from Cell Signaling (Danvers, MA). mAb to the catalytic subunit of calpain-1 was from Sigma–Aldrich (St. Louis, MO, U.S.A.).
The meningothelial meningioma samples were obtained from the Neurosurgery Department of the IRCCS-AOU San Martino IST, (Genova, Italy) after patients’ informed consent and Institutional Ethical Committee approval. The patients underwent surgery for the first time and never received chemo- or radiotherapy. Pathological analysis classified meningothelial meningiomas as grade I according to World Health Organization criteria [24 (link)]. The tissue specimens collected in the operation room were quickly frozen in liquid nitrogen, and stored at −80°C until processing.

Triton^® X-100 was purchased from Sigma. Protein G-sepharose 4 Fast Flow, nitrocellulose membrane and ECL SELECT^® Detection System were obtained from GE Healthcare. Mouse anti-A2A primary antibody was purchased from Merck Millipore Corporation (05-717; [45 (link)]); goat anti-GFAP primary antibody was purchased from Santa Cruz Biotechnoloy Inc; rabbit anti-A2A and anti-D2 primary antibodies were purchased from Alomone Labs. The anti-rabbit secondary antibody and the Protease Inhibitor Cocktail were obtained from Cell Signaling Technology (Leiden, The Netherlands).