Protein kinase b akt

The protein content was determined using bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). About 60-80 µg of protein from the lysates were loaded and fractionated on SDS-PAGE, transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK) and detected with antibodies against P-gp (GeneTex, Inc. Irvine, CA, USA), the mammalian target of rapamycin (mTOR) (Cell Signaling, Danvers, MA, USA), phosph-mTOR (Cell Signaling), protein kinase B (AKT) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), phosph-AKT (Santa Cruz Biotechnology, Inc.), p70s6K (Cell Signaling), phosph-p70s6K (Cell Signaling), caspase 3 (Cell Signaling), β-actin (Sigma Aldrich). Rabbit anti-mouse IgG-peroxidase antibody (Sigma Aldrich) and goat anti-rabbit IgG-peroxidase antibody (Sigma Aldrich) were used as the secondary antibody. The protein-antibody complexes were visualized using enhanced chemiluminescence system. The Western blotting signals were quantified with ImageJ software (rsbweb.nih.gov/ij/) 20 (link).

The protein content was used a bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA) to determine the protein concentration. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to fractionate protein samples. The protein samples were transfer to the hybond-enhanced chemiluminescence nitrocellulose membranes (Pall Life Science, Glen Cove, NY, USA). The membranes were incubated with various antibodies, including CXCR4 (GeneTex Inc. Irvine, CA, USA), the protein kinase B (AKT) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylation-AKT (Santa Cruz Biotechnology), mammalian targets of rapamycin (mTOR) (Cell Signaling, Danvers, MA, USA), phosphorylation-mTOR (Cell Signaling), p70 ribosomal S6 kinase (p-p70S6K) (Cell Signaling), phosphorylation-p70S6K (Cell Signaling), and β-actin (Sigma-Aldrich). The appropriate horseradish-peroxidase-conjugated secondary antibodies were used and enhanced chemiluminescence system (T-Pro Biotechnology, New Taipei City, Taiwan) to detect the protein-antibody complexes. Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA) was used to transfect with the constitutively active AKT plasmids to cells.