Ez ecl enhanced chemiluminescence detection kit

Primary antibodies: mouse anti-PARP-1, rabbit anti-phospho-Ser473 Akt, rabbit anti-phospho-Thr308 Akt, rabbit anti-Akt, rabbit anti-phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti-phospho-MEK1/2, mouse anti-MEK1/2, mouse anti-p21waf1, rabbit anti-p27, were from Cell Signaling Technologies (Boston, MA, USA). Mouse anti-human GAPDH and rabbit anti-cyclin A were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies: horseradish peroxidase (HRP)—conjugated goat anti-rabbit and goat anti-mouse IgG (H + L) antibodies were from Jackson (Baltimore Pike West Grove, PA, USA). EZ-ECL enhanced chemiluminescence detection kit, RPMI1640 medium, L-glutamine, fetal bovine serum, trypsin, antibiotics and phosphate buffered saline (PBS) were from Biological Industries (Beit-Ha-Emek, Israel). Propidium iodide, phosphatase inhibitor cocktails 2 and 3, sulphorodamine B (SRB), trichloroacetic acid and acetic acid were from Sigma Aldrich (St. Louis, MO, USA). ZSTK474, Selumetinib and AEW-541 were from Selleckchem (Houston, TX, USA). Complete mini protease inhibitor cocktail, RNAse A and Triton X-100 were from Roche Diagnostics Gmbl (Mannheim, Germany).

Detection of ovarian M1, M3, and M5 and the internal control β‐actin by western blot was performed, following separation by SDS–PAGE on 10% polyacrylamide gels under reducing conditions. The proteins were transferred to nitrocellulose membranes (pore size: 0.45 μm; Schleicher & Schuell), blocked with 5% milk for 1 h, and probed with either rabbit polyclonal anti‐M1 antibody (AMR‐001, Alomone Labs) in a 1:1000 dilution, rabbit polyclonal anti‐M5 antibody (AMR‐005, Alomone Labs) in a 1:500 dilution, overnight or in a 1:10,000 dilution of mouse monoclonal anti‐β‐actin (A1978, Sigma–Aldrich Co.) for 1h. The antibody complexes were detected using goat antirabbit IgG Fc (HRP) (ab97200, Abcam, Inc.) or horse antimouse IgG Fc (HRP) (BA‐2000, Vector Lab) both at 1:10000, and for chemiluminescence, an EZ‐ECL Enhanced Chemiluminescence Detection Kit (Biological Industries, KBH, Israel) was used. Chemiluminescence was captured using a G‐Box Syngene system (Syngene Headquarters). Band intensity was quantified by ImageJ software (Rasband, 1997–2014), and values obtained were normalized to β‐actin. Each protein sample detection was assessed in three independent experiments.

U-87 MG cells were treated with the indicated concentrations of D-Δ(1-18)N-Ter-Antp peptide in serum-free DMEM for 6 h. Then cells were lysed in lysis buffer (100 mM Tris/HCl pH 8.0, 5% sodium dodecyl sulfate [SDS]), supplemented with a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). The lysates were vortexed and heated at 70 °C for 10 min. Finally, cell lysates were centrifuged (15,000× g, 10 min at 4 °C), and the protein concentration of the supernatant was determined according to the Lowry assay [32 (link)] with a slight modification. Protein samples were stored at −80 °C until subsequent gel electrophoresis. Protein samples (20 μg) were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted using selected primary antibodies (sources and dilutions are listed in Table S1), followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Table S1). Proteins were detected using the EZ-ECL Enhanced Chemiluminescence Detection Kit (Biological Industries, Beit Haemek, Israel). Band intensities were analyzed with densitometry using FUSION-FX software (Vilber Lourmat, Collegien, France), and the values were normalized to β-actin.