Western Blot Analysis of AKT Signaling

Cells were washed by PBS for 3 times and then lysed in RIPA lysis buffer (Wuhan Servicebio Technology Co., Ltd.) with the protein concentration measured by a Pierce™ Microplate BCA Protein Assay Kit (Invitrogen, Carlsbad, CA, USA) [47 (link)–49 (link)]. Next, equal amounts of total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membrane and followed by the incubation in 5% skimmed milk at room temperature for additional 1 h to block the nonspecific binding of primary antibodies. After that, the membranes were incubated with indicated primary antibodies at 4°C overnight and the secondary antibody at room temperature for additional 1 h on the second day. Finally, the bands were visualized using electrochemiluminescence reagent and analyzed by Image Lab software (Version 6.0, Bio-Rad). The primary antibodies against phospho-KT (p-AKT), total-AKT (t-AKT), PTEN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were all purchased from Cell Signaling Technology (Danvers, MA, USA).