PC12 cells were incubated at 37°C for 24 hours with vehicle control (0 nM) and 1 μM of Galanin (1-15) (porcine, rat) (ab 141152). Decreased expression of AKT1 (phospho S473) (ab81283) in PC12 cells correlates with an increase in Galanin (1-15) (porcine, rat) concentration, as described in literature.Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 30μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab81283 at 1 μg/ml and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.