Anti akt 4691

The protein samples were extracted from different groups of cells by using cracking buffer solution RIPA (Solarbio, Beijing, China) at 4 °C for 15 min. The supernatants were centrifuged at 15,000 rpm, and the total protein was quantified by the BCA Kit (Solarbio). Thereafter, the protein samples were separated on 10% SDS-polyacrylamide gels and transferred to 0.45 μm PVDF membranes (Millipore, Billerica, MA). The blots were blocked with 5% Bovine Serum Albumin (BSA) in PBS buffer for 1 h, and then incubated with the appropriate primary antibodies (1:2000) at 4 °C overnight. After washing, the blots were incubated with the corresponding secondary antibodies (1:5000) at room temperature for 2 h. The signals were visualized by using a SuperSignal West Pico Substrate Kit (Pierce, Thermo Fisher Scientific), and analysed by using ImageJ software (National Institutes of Health, Bethesda, MD). The anti-Hexokinase 2 (22029-I-AP) and anti-STAT3 (10253–2-AP) antibodies were obtained from ProteinTech Group Inc. (Rosemont, IL), the anti-p-AKT (#4060) and anti-AKT (#4691) antibodies were purchased from Cell Signaling Technology (Danvers, MA), anti-p-STAT3 (BM4835) and anti-GAPDH (BM1623) antibodies, and all secondary antibodies were from Boster Biological Technology Ltd. (Wuhan, China).

GST and GST-p37 fusion proteins were expressed and purified by our lab. AG1478 was purchased from Sigma-Aldrich (St Louis, MO, US) and wortmannin was purchased from Cell Signaling (Danvers, MA, US). Both of them were dissolved in DMSO, aliquoted and stored at −20°C. Anti-phospho-EGFR (Y1068) (#2234), anti-phospho-PI3K (S458) (#4228), anti-phospho-AKT (S473) (#4060), anti-PI3K (#4257), and anti-AKT (#4691) antibodies were purchased from Cell Signaling. Anti-AKT antibody (BS1784) was purchased from Bioworld (St. Louis Park, MN, US). Anti-p37 monoclonal antibody PD4 was generated in our lab [27 ].