Western Blot Analysis of Apelin Signaling

Western blot was performed as previously described 26 (link). Briefly, the total protein was loaded to a SDS-PAGE gel (6% stacking gel and 15% separating gel). After gel electrophoresis, the proteins were transferred to a nitrocellulose membrane at 70V for 2 h and then blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. Next the blocked membrane was incubated with the primary antibody overnight at 4 °C. Primary antibodies used were APLN (sc-33804), APLNR (sc-33823) and β-actin (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA); Phospho-PI3 Kinase p85 (Tyr458) (4228), Akt (9272), phospho-Akt (Ser473) (9271), GSK3β (9315), phospho-GSK3β (Ser9) (9336), Cleaved Caspase-3 (9661), Cleaved PARP (5625) and Cyclin D1 (2922) (Cell Signaling Technology, Danvers, MA). Protein quantification was performed by Image J.