Sds polyacrylamide pre cast gels

For immunoblot analysis, whole cell lysates were prepared according standard procedures, 20–24 h after splitting the culture. Samples equivalent to 20µg of protein were separated using 4–12% SDS-polyacrylamide pre-cast gels (Invitrogen, Karlsruhe, Germany) and transferred to nitrocellulose membranes according to manufacturer’s prescriptions. For protein detection, membranes were incubated with respective primary and species-specific peroxidase-labeled secondary antibodies according to standard protocols. The levels of protein expression were quantified using Image J program and normalized to the β-actin levels.
The primary antibodies used were: rabbit polyclonal anti-PTEN, rabbit polyclonal anti-PI3K p110, mouse monoclonal anti-phospho-AKT (Ser473), rabbit monoclonal anti-phospho-mTOR (Ser2448) (all from Cell Signaling, Danvers, MA), mouse monoclonal anti-p53 (Merck Chemicals Ltd., Nottingham, UK), mouse monoclonal anti-Fatty Acid Synthase (BD Biosciences, Heidelberg, Germany), mouse monoclonal anti-MDM2 (SMP14) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), mouse monoclonal anti-β-actin (Sigma, Deisenhofen, Germany). Secondary species-specific antibodies for Western blot were labelled with horseradish-peroxidase (DAKO, Hamburg, Germany).

The expression of marker proteins was tested using western blotting as previously described [24 (link)]. Cellular lysates were prepared 30 min and 24 h post-IR according to standard procedures. Forty μg of protein per lane were separated using 4–12% SDS-polyacrylamide pre-cast gels (Invitrogen, Karlsruhe, Germany) and transferred to nitrocellulose membranes according to the manufacturer’s prescriptions. The levels of protein expression were quantified using Image J (Wayne Rasband, National Institutes of Health, Bethesda, MD) and normalized to β-actin intensity. Experiments were repeated at least three times, unless otherwise noted.

For immunoblot assays, whole-cell lysates were prepared 30 min and 24 h post-IR, according to standard procedures. Samples equivalent to 40 μg of protein were separated using 4–12% SDS-polyacrylamide pre-cast gels (Invitrogen, Karlsruhe, Germany) and transferred to nitrocellulose membranes. For protein detection, membranes were incubated with respective primary and species-specific peroxidase-labeled secondary antibodies according to standard protocols. The levels of protein expression were quantified using the software ImageJ (NIH, Bethesda, MD) and normalized to β-actin levels.