Anti phospho akt rabbit monoclonal antibody

The Western blot was performed as described earlier (Mali et al., 2016b ; Pan et al., 2016 ). In brief, protein samples from cardiac mitochondria and muscle plasma membrane fractions were separated on SDS-polyacrylamide gels by electrophoresis. The proteins were then transferred to immobilon-P membranes (Millipore, Billerica, MA). Levels of 4HNE-protein adducts in cardiac mitochondrial samples were determined using antibodies of anti-4HNE-Cys/His/Lys rabbit antibody (Millipore) along with anti-aconitase mouse monoclonal antibody (Abcam) was used to detect aconitase, a housekeeping marker, for comparison. In the proteins from plasma membrane fractions of muscle, we measured phospho-AKT using anti-phospho-AKT rabbit monoclonal antibody (Cell Signaling Technology), AKT using AKT rabbit polyclonal antibody (Cell Signaling Technology), phospho-AS160 using anti-phospho-AS160 (Thr642) rabbit polyclonal antibody (Cell Signaling Technology), AS160 using anti-AS160 rabbit polyclonal antibody (Cell Signaling Technology). Anti-Na⁺K⁺ATPase rabbit polyclonal antibody (Cell Signaling Technology) and anti- α-sarcomeric actin goat polyclonal antibody (Santacruz Biotech) were used as a loading controls. The bound primary antibodies were added to horseradish peroxidase (HRP)-coupled respective secondary antibodies, and then visualized by chemiluminescence detection reagents.