Radioimmunoprecipitation assay (ripa)

After lysis with RIPA (Epizyme, Shanghai, China), the proteins were extracted and centrifuged. The supernatants were collected, and the total protein concentrations were measured using BCA (Epizyme, Shanghai, China) assay. Proteins were separated using SDS-PAGE (Epizyme, Shanghai, China) and transferred to PVDF membranes (Millipore, Germany) and blocked with protein free rapid blocking buffer for 1 h. The membranes were immunoblotted with the target primary antibodies at 4°C overnight. After washing with TBST, the membranes were treated with HRP-conjugated secondary antibody at room temperature for 1 h. Bands were visualized using chemiluminescence, and gray value analysis of the bands was performed using Image J software. The manufacturer and catalog of antibodies we used were listed in the Supplementary Table 1.

NCI-H1703, NCI-H226 and SK-MES-1 cells were collected following transfection with siRNA-EIF4G1. The cells were lysed using RIPA (EpiZyme) with protease inhibitor cocktails (Bimake). A total of 30 μg of cell lysate was added to 6–20% SDS-PAGE gels and the proteins were transferred to 0.45 mm PVDF membranes (PerkinElmer Life Sciences, Inc.). The membranes were blocked in TBST with 5% BSA for 2 h at room temperature and they were subsequently incubated with primary antibodies against EIF4G1 (Cell Signaling Technology #8701, 1:1,000), AKT (Immunoway #YP0007, 1:1,000), p-AKT (ABclonal technology #AP1208, 1:1,000), mTOR (Bioss Anti Bodies #bs3494R, 1:1,000), cyclin D1 (ABclonal technology #A11022, 1:1,000), β-actin (Cell Signaling Technology, 1:1,000) at 4 ℃ overnight (more than 12 h). HRP (Invitrogen) was conjugated to a secondary antibody which was incubated at room temperature for 1 h with the membranes. Enhanced chemiluminescence reaction (Beyotime Institute of Biotechnology) was used to identify protein bands, which were visualized using autoradiography.

The total protein was extracted with RIPA (EpiZyme, Shanghai, China), according to the manufacturer’s instruction. Then, the protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was incubated overnight with specific antibodies at 1:1000 at 4 °C. The primary antibodies used were β-actin (#3700), phosphorylated (p)-mTOR (Ser2448) (# 5536S), total (t)-mTOR (#2983) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) (#2435 T) (Cell Signaling Technology, MA, US). The chemiluminescent assay kit (EpiZyme, Shanghai, China) was used for color reaction.