Quantity one 4.5 tool

Protein extracts (30 μg) from each of 8 NSCLC and cancer-free lung tissue were incubated in Laemmli buffer with DTT, resolved on SDS-PAGE and then transferred onto nitrocellulose membranes (GE Healthcare, TX, USA) by Mini trans-blot electrophoresis transfer as previously described (Bio-Rad Laboratories, CA, USA) [23 (link)]. p-ERK1/2, ERK1/2, p-AMPK, AMPK, p-AKT, AKT, p-P38, P38, p-IKBα, IKBα antibodies were from Cell Signaling, Netherlands; NF-κβ antibody was from BD bioscience; CAI antibody was from Santa Cruz Biotechnology, MA, USA; CAII antibody was from Rockland, PA, USA; GAPDH and β-actin antibodies were from Sigma-Aldrich, MO, USA. Immunoblots were detected using the ECL-Advance Western Blotting Detection kit (GE Healthcare, TX, USA). Western blot images were scanned by PDquest 7.1 software (Bio-Rad Laboratories, CA, USA). Densitometric measurements were made with the Quantity One 4.5 tool (Bio-Rad Laboratories, CA, USA). Each experiment was performed at least three times in duplicate.

Protein extracts were electrophoresed on polyacrylamide gel (Invitrogen) and transferred onto nitrocellulose membranes (Millipore). After 1 hour (h) blocking with 5% dry fat milk in phosphate-buffered saline (PBS) containing 0.02% Tween-20, the membranes were incubated with the primary antibody overnight at 4 C° and with the secondary antibody for 1h at room temperature. Primary antibodies used are: anti-human BARD1 (cod-A300-263A, Bethyl, 1:1000), γH2AX (phosphoSer139) (cod-H5912, Sigma Aldrich, 1:1000), phosphor-p53(Ser-15) (cod-9284 Cell Signaling, 1:500), p53 (sc-6243, Santa Cruz, 1:500), Cyclin B (sc-752 Santa Cruz, 1:500), CDK1 (sc-54, Santa Cruz, 1:1000); phospho-H3 (06-570 Millipore). Mouse monoclonal anti-β-Actin antibody (cod-A5441, Sigma-Aldrich, 1:6000) and anti-H3 (cod-06-755, Millipore, 1:1000), were used as loading control for cytosol and nuclei extracts respectively. Secondary peroxidase-labeled antibody to rabbit IgG (cod041506, KPL) and to mouse IgG (cod041806, KPL) were diluted at 1:2000. Protein bands were visualized with enhanced chemiluminescence plus reagent (GE Healthcare). The protein bands image were acquired with GelDoc 2000 system (Bio-Rad) and the densitometry measurement was performed by Quantity One 4.5 tool (Bio-Rad).