α caspase 8

Protein expression and phosphorylation status was detected by Western blot analysis. The antibodies α-S6K T389, α-4EBP1 T37/46, α-Bax, α-caspase-8, α-Bcl-2, α-Bcl-x_L, α-CDK1 T161, α-CDK1 Y15, α-MEK1/2 S217/221 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibodies α-GAPDH, α-Mcl-1, α-Bak were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). α-caspase-9 and α-caspase-3 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). The antibodies α-p53, α-Cyclin B1, and α-Cyclin A were obtained from BD Pharmingen (Heidelberg, Germany). The antibodies α-pHH3 and α-γH2AX were purchased from Sigma-Aldrich (Taufkirchen, Germany). Treated cells were lysed in RIPA buffer^{67 (link)}, proteins were isolated by centrifugation and protein concentration was measured using the BCA method. SDS-PAGE was performed as described previously^{68 (link)}. Transfer of proteins onto nitrocellulose membrane was performed with Towbin buffer and wet blot using the Trans-Blot® System from Bio-Rad Laboratories (Hercules, CA, USA). For immunodetection, membranes were incubated with primary antibody at 4 °C overnight. Radish peroxidase labeled secondary antibody was added for 2 h at room temperature and chemiluminescence signal was detected by the use of the ChemiDoc™ MP System (Bio-Rad Laboratories, Hercules, CA, USA).