Anti phospho akt ser473 rabbit mab

Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and stored in aliquots at 20°C until use. Twenty micrograms of cell lysates were mixed with an equal volume of Laemmli sample buffer, denatured by boiling, and separated by SDS-PAGE. The separated proteins were then transferred to a nitrocellulose membrane (BioRad). The membranes were blocked using 5% non-fat dry milk for 1 h at room temperature and probed with anti-phospho-Akt (Ser473) rabbit mAb (Cell signaling, Beverly, MA), anti-phospho-RhoA (Ser188) rabbit mAb (Cell signaling), anti-phospho-Myosin light chain (Ser19) rabbit mAb (Cell signaling), anti-phospho-FAK (Tyr925) rabbit mAb (Cell signaling), anti-Akt rabbit mAb (Cell signaling), and anti-phospho-CXCR4 (Ser339) rabbit mAb (Sigma-Aldrich, St. Louis, MO). After washing three times in TBST, the blots were incubated with IgG horseradish peroxidase conjugated anti–rabbit antibody (Cell signaling, Beverly, MA) at a final concentration of 1:5000 for 2 h at room temperature. Immunoblots were developed using the enhanced chemiluminescence (ECL) reagent (Cell signaling, Beverly, MA) and visualized using FluroChemQ processor (Proteinsimple, Santa Clara, CA).