Antibodies against p akt

3-MA, rapamycin, Chloroquine, compound C and Bafilomycin A1 were purchased from Sigma Aldrich (St. Louis, MO, USA). DMEM, Opti-MEM, penicillin-streptomycin, antibody against glyceraldehyde−3-phosphate dehydrogenase (GAPDH), AMPK siRNA, and siRNA Transfection Reagent were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against LC3, p62 and LAMP2 were bought from Abcam (Cambridge, MA, USA), the antibodies against p-AKT, AKT, AMPK, p-AMPK, mTOR and p-mTOR were from Cell Signaling Technology (Danvers, MA, USA), the recombinant active full-length human Akt1 protein (rAkt1) was purchased from Abcam (Cambridge, MA, USA).

The proteins from ARPE-19, Y79, and WERI-Rb-1 cells were extracted using RIPA buffer (Beyotime). The concentration was determined by a BCA kit. Equal amounts of the total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, the membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 2 hours. Then, the membranes were washed with TBST and subjected to chemiluminescence analysis using the ECL-Plus kit (Amersham Biosciences). The antibody against RRS1 was from Abcam. Antibodies against p-AKT (1 : 1000), AKT (1 : 1000), p-mTOR (1 : 1000), and mTOR (1 : 1000) were from Cell Signaling Technology. GAPDH primary antibody (1 : 10000) and all the secondary antibodies (1 : 10000) were from Proteintech.

Cell proteins were extracted with RIPA buffer containing protease and phosphatase inhibitors. After 80–120 V electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane (Invitrogen). After blocking with 5% skim milk, the membrane was incubated with antibodies. Antibodies against p-AKT, AKT, p-ERK, and ERK were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against KRAS, E-cadherin, N-cadherin, Vimentin, ZEB2 and β-actin were supplied by Proteintech Group (Wuhan, China). An antibody against MT-CO1 was purchased from ABclonal (Wuhan, China). Protein bands were visualized with an enhanced chemiluminescence (ECL) reagent (Pierce, Rockford, IL, USA).