Western blot and human apoptosis antibody array-membrane

Cells were washed with cold PBS and lysed in ice-cold lysis buffers containing Western, IP lysate and PMSF. Total protein concentrations were determined using the BCA method with BCA Protein Assay Kit (HyClone-Pierce, Logan, UT, USA). Proteins (20 μg per lane) were separated by electrophoresis on a 10% SDS-PAGE gel, and transferred to nitrocellulose membranes. The membranes were blocked with TBST solution containing 5% skimmed milk for 1 h. Primary antibodies specific to Akt (1:1000), p-Akt (1:1000), Cyclin D1 (1:2000), CDK6 (1:1000), p21 (1:500) were added and incubated at 4 °C overnight. Then the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:3000) for 1 h at room temperature. Western Chemiluminescent HRP Substrote kit was used for coloring (Millipore, Schwalbach, Germany). GAPDH served as the internal standard and the blot bands were visualized with enhanced chemiluminescence (ECL) (Amersham, Chicago, IL, USA) system.

For human apoptosis antibody array, total protein from was PC-3 in shCtrl and shPSMC2 groups were collected after fully lysed by lysis buffer. Protein samples were added for incubating with blocked array antibody membrane overnight at 4 °C. After washing, 1:100 Detection Antibody Cocktail was added incubating for 1 h, followed by incubated with HRP linked streptavidin conjugate for 1 h. All spots were visualized by enhanced chemiluminescence and the signal densities were analyzed with ImageJ software (National Institute of Health, Bethesda, MD, USA).