P bad ser136

RTECs were lysed in RIPA buffer for 10 minutes on ice and then subjected to sonication for 10s. The lysate was centrifuged at 14,000 g for 10 min at 4°C. The supernatant was collected and protein concentration was determined by Bradford assay. Equal amounts of protein were separated on SDS-PAGE, transferred onto a nitrocellulose membrane, blocked, and incubated with primary antibodies of cleaved caspase-3 (Cell Signaling, 9661), XIAP (Cell Signaling, 2042), Akt1 (Cell Signaling, 2938) pAkt Ser473 (Cell Signaling, 9271), or pBAD Ser136 (Cell Signaling, 4366) overnight at 4°C. After washing, the membrane was incubated with a horseradish peroxidase-labeled secondary antibody. ECL was used as a method of detection. Each membrane was stripped and reprobed with anti β-actin antibody β-actin (Cell Signaling, 4970) to verify equal protein loading. Immunoblots for every protein were performed on samples from 4 separate, independent experiments for each condition to ensure reproducibility. To quantitate the results, densitometry and statistical analyses were performed. The densitometry values were adjusted to loading control (β-actin).