Antibodies against akt and phospho akt ser473

Sulforhodamine B (SRB) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Antibodies against AKT and phospho-AKT (Ser473) were obtained from Cell Signaling Technology (MA, USA). Antibodies against Bax and Bcl-2 were from BD Pharmigen (BD Biosciences, San Josè, CA, USA). Antibodies against ERK1/2 (C-14), phospho-ERK (E-4), PARP-1, and p53 (DO-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-ErbB2 antiserum was provided by Dr. M.H. Kraus (University of Alabama, Birmingham, USA). A rabbit polyclonal antibody against actin and goat anti-mouse or anti-rabbit IgG peroxidase-conjugated secondary antibodies were from Sigma-Aldrich.

Western blot for phospho-AKT (Ser473), total AKT, and β-actin were performed on gWAT protein extracts as described [45 (link)]. Briefly, the protein concentration was measured using the DC protein assay (Bio-Rad laboratories, Veenendaal, The Netherlands), and 20 µg of a protein sample was run on a 12% acrylamide gel and blotted on a PVDF membrane (Merck Millipore, Amsterdam, The Netherlands). The membrane was incubated with primary antibodies (the antibody against β-actin was purchased from Abcam, Cambridge, UK; antibodies against AKT and phospho-AKT (Ser473) were purchased from Cell Signaling Technology, via BIOKÉ, Leiden, The Netherlands) at 4 °C overnight and then was incubated with goat anti-mouse secondary antibody for β-actin or donkey anti-rabbit secondary antibody (LI-COR, Lincoln, NE, USA) for the other antibodies, all at room temperature for 1 h. The membrane was scanned on an Odyssey scanner (LI-COR, Lincoln, NE, USA). Bands were analyzed using Odyssey software V3.0 (LI-COR, Lincoln, NE, USA).