Westar supernova

Cells were harvested and lysed in a buffer containing proteases and phosphatases inhibitors. For preparation of cytosolic and nuclear extracts, cells were washed with phosphate-buffered saline (PBS) and the pellet was resuspended in a low salt buffer and spun (the supernatant is the cytosolic fraction). Nuclei-containing pellets were resuspended in high salt buffer. Protein concentration was determined using the Bradford reagent. Samples were electrophoresed through 10 or 12% SDS–PAGE, followed by protein transfer to nitrocellulose membranes. Blots were blocked with 5% skim milk and incubated with antibodies against: phospho-IGF1R/INSR (#3024), IGF1R β-subunit (#3027), INSR β-subunit (#3025), phospho-AKT (#9271), AKT (#9272), phospho-ERK1/2 (#9106) and ERK1/2 (#9102). Antibodies were from Cell Signaling Technology. An antibody against lamin B was obtained from Abcam (Cambridge, UK) and anti-heat shock cognate (HSC70) (#Hsp73) was from Santa Cruz Biotechnology (Dallas, TX, USA). Blots were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the enhanced chemiluminiscence reaction (Westar Supernova, Cyanagen, Bologna, Italy). HSC70 was used as a loading control.

Cells were washed with ice-cold phosphate-buffered saline (PBS), lysed in buffer contained 50 mM TRIS-HCl, pH 7.5, 400 mM NaCl, 10% glycerol, 0.5% NP40, 1% Tryton X-100 1 mM EDTA, 1 mM EGTA 2 mM DTT. All reagents were from Sigma-Aldrich. Buffer were supplemented with a protease inhibitor cocktail (P1860-Sigma-Aldrich) and phosphatase inhibitors cocktails (Sigma-Aldrich P5726 and Sigma-Aldrich P0044). Aliquots (20–60 µg) from total cell lysate proteins were resolved on 8–15% SDS–PAGE gels and analysed by immunoblotting with the indicated antibodies followed by decoration with peroxidase-labelled anti-rabbit (Thermo Fisher Scientific) or anti-mouse immunoglobulin G (IgG) (Dako-Agilent, Santa Clara, CA, USA) respectively. Blots were developed with enhanced chemiluminescence (ECL) Westar Supernova (Cyanagen, Bologna, Italy), following the instructions of the manufacturers.

Cells were harvested in RIPA buffer (150 mM sodium chloride, 1% Triton-X, 50 mM Tris, pH 8.0, 10 mM EDTA, pH 13, 10 mM PMSF, PIC). Protein quantification was performed using the Protein Assay Bicinchoninate Kit (Nacalai Tesque, Japan) and 2 mg/ml BSA (Thermo Fisher Scientific) was used to construct the standard curve. The purified protein lysates were subjected to SDS-PAGE followed by Western Blotting. The blots were developed using the WesternBright enhanced chemiluminescent HRP substrate (Advansta, CA, United States) or Westar Supernova (Cyanagen, Italy) and the bands were captured with the ChemiDoc XRS imager (BioRad).

Proteins from the parenchymal region of each mammary gland were extracted with RIPA buffer containing 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 5 mM EDTA (Sigma), protease inhibitor cocktail and phosphatase inhibitor cocktail II (both from Sigma). Equal amounts of protein, determined by QPRO-BCA Kit Standard (Cyanagen, Bologna, Italy) according to the manufacturer’s protocol, were subjected to SDS–PAGE. Fractionated proteins were transferred to nitrocellulose filters and the uploading of equal amounts of protein per sample was confirmed by staining the blot with Ponceau S (Sigma). The expression levels of phosphorylated S6 (pS6) and β-actin were determined according to the signals generated with their respective antibodies. Signals were generated with WESTAR Supernova (Cyanagen, Bologna, Italy) combined with Super Signal chemiluminescent substrate (Thermo Fisher Scientific), and detected in a Syngene analyzer (Cambridge, UK).

Confluent cells were washed with ice-cold phosphate-buffered saline (PBS) containing 5 mM EDTA and centrifuged at 1100 rpm. After discarding the supernatant, lysis buffer was added and the cells were incubated on ice for 10 min. Cells were then centrifuged at 13000 rpm for 10 min. Protein concentration was determined by the Bradford method. Samples were electrophoresed through 10% SDS-PAGE, followed by blotting of the proteins onto nitrocellulose membranes. After blocking with 5% skim milk, the blots were incubated overnight with antibodies against ZYG11A (Abcam plc, Cambridge, UK) and heat shock cognate protein 70 (#Hsp73, Sigma-Aldrich). In addition, antibodies against pTEN (#9559), cyclin D1 (#2926) and p21 Waf1/Cip1 (#2947) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p53 (mixture of DO-1 and Pab 1801) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After incubation, the blots were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the enhanced chemiluminescence reaction Westar Supernova (Cyanagen, Bologna, Italy]. HSP70 was used as a loading control.

Cells were harvested and lysed in a buffer containing proteases and phosphatases inhibitors. Samples were electrophoresed through 5, 10, 12, or 15% SDS–PAGE, followed by protein transfer onto nitrocellulose membranes. Blots were blocked with 5% skim milk and incubated overnight with antibodies against Cyclin D1 (#DCS6), ATM (#D2E2), JAK1 (#3332), STAT3 (#79D7), Caspase-3 (#8G10), AKT (#9272), mTOR (#7C10), and Ampk (#2532). These antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). An antibody against SOD2 was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Antibodies against Chek2 (#A-11), p53 (mixture of DO-1 and 1801), and Hsp70 (#B-6) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). An antibody against Ras (#Ab-3) was purchased from Oncogene Research Products (La Jolla, CA, USA). Blots were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the enhanced chemiluminiscence reaction (Westar Supernova, Cyanagen, Bologna, Italy). Hsp70 was used as a loading control.