Htra2 omi

Cells were harvested by washing with ice-cold lysis buffer (250 mM sucrose, 1.5 mM MgCl2, 10 mM KCl, 20 mM HEPES 7.6 pH, 1 mM of dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 10 mM sodium orthovanadate, 20 mM sodium pyrophosphate, and protease inhibitors) for 15 min then supplemented with 5% of Nonidet P-40 for 2 min. Cytosolic fraction was collected from the supernatant after centrifugation at 14,000g for 10 min. Bradford reagent (Bio-Rad Laboratories Hercules, CA) was used to determine the protein concentrations, and equal amounts of samples loaded onto 10–15% SDS-polyacrylamide gels were separated by electrophoresis and transferred to PVDF membranes. Membranes were blocked and incubated with PAR-1 (Abcam, Cam- bridge, MA), cofilin, Caspase3, HtrA2/omi, or NF-κB (Cell Signaling Technology, Danvers, MA) primary antibodies at 4° C overnight. After washing, membranes were incubated for 1h at room temperature with a horseradish peroxidase-conjugated secondary antibody (1:5000; Jackson ImmunoResearch, West Grove, PA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or beta-actin were used as loading controls, and band intensity was analyzed using Bio-Rad Chemi-Doc XRS Image Lab Software.