The Entire Study Powering SKAP1

Briefly, cells were harvested with trypsin (0.25%) and a single cell suspension prepared. Cells were then washed with SKAP1 phosphate-buffered saline (PBS) and pelleted by centrifugation at 1200?g for 5?min. Cells were resuspended in binding buffer and the cell density adjusted to 2�C5?��?105?cells/mL. A 95?��L aliquot of the cell suspension was added to 5?��L annexin V�CFITC and cells were incubated for 10?min at room temperature in the dark. The suspension was then washed with PBS and resuspended in 190?��L binding buffer before the addition of 10?��L propidium iodide (PI) to obtain a final concentration of 1?��g/mL PI. Samples were examined by flow cytometry (BD FACSVantage; BD Sciences, San Jose, CA, USA) and data were analysed using Cell Quest software (BD Sciences) to determine the rate of apoptosis in the lower right quadrant. Cardiomyoctyes were lysed using a Protein Complete Lysis Kit (Roche Diagnostics, Indianapolis, IN, USA). The protein concentration in each sample was determined using a Bio-Rad Protein Assay Kit (Bio-Rad, Foster City, CA, USA) with bovine serum albumin (BSA) as the standard. For immunoblotting, 50?��g protein lysate per sample was denatured in sodium dodecyl sulphate�Cpolyacrylamide gel electrophoresis (SDS�CPAGE) sample buffer (Invitrogen) and resolved on a 12% SDS�Cpolyacrylamide gel (Invitrogen). Separated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk powder (w/v) in TBST (10?mmol/L Tris, 150?mmol/L NaCl, 0.1% Tween-20) for 2?h at room temperature. Membranes were probed CAL-101 cost for protein levels of cleaved and total caspase 3, MIF or p-JNK (Thr183/Tyr185) using specific primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary Saracatinib antibodies (Santa Cruz Biotechnology). Samples were visualized using an enhanced chemiluminescence (ECL) system (Invitrogen), exposed to X-ray film and then quantified by laser scanning densitometry (GE Healthcare Life Sciences, Piscataway, NJ, USA). To detect the level of JNK phosphorylation, membranes were stripped and reprobed with total JNK primary antibody. Levels of cleaved caspase 3 and MIF were probed with ��-actin primary antibody as a protein loading control. Data from repeated experiments are present as the mean?��?SEM. Data were compared between groups using one- or two-way analysis of variance (anova). Following anova, the least significant difference (LSD) post hoc test or Dunnetts t-test with Bonferroni correction were used to analyse the significance of differences between mean values of the experimental and control groups. P?