Samples of protein fraction containing equal amounts of protein per lane (40 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For Western blot analysis, separated proteins were transferred from gel to a nitrocellulose membrane. The quality of the transfer was controlled by Ponceau S staining of nitrocellulose membranes after the transfer and protein loading by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeper. Specific anti-Akt kinase, anti-Bcl-2 and anti-BAX (from Santa Cruz Biotechnology) and antiphospho (P)-Akt kinase (from Cell Signaling Technology) antibodies were used for the primary immunodetection.
Peroxidase-labelled anti-rabbit immunoglobulin (Cell Signaling Technology) was used as the secondary antibody. Bound antibodies were detected by the enhanced chemiluminiscence detection method using Carestream PC program. Protein levels of Akt, P-Akt and BAX were normalized to GAPDH. PI3K/Akt activity was expressed as a ratio of P-Akt and Akt. ColorBurst marker (Sigma Aldrich) was used for quantitative molecular mass determination of transferred proteins.
Peroxidase-labelled anti-rabbit immunoglobulin (Cell Signaling Technology) was used as the secondary antibody. Bound antibodies were detected by the enhanced chemiluminiscence detection method using Carestream PC program. Protein levels of Akt, P-Akt and BAX were normalized to GAPDH. PI3K/Akt activity was expressed as a ratio of P-Akt and Akt. ColorBurst marker (Sigma Aldrich) was used for quantitative molecular mass determination of transferred proteins.