Eif4g1

NPC cells with indicated transfection were lysed in radio-immunoprecipitation assay (RIPA) lysis buffer (Santa Cruz, Dallas, TX, USA) for half an hour on ice. Excised xenograft tumors were homogenized and lysed in RIPA lysis buffer (Santa Cruz) for 1 hon ice. The supernatants were harvested after centrifugation at 10,000 g for 15 min. Protein was quantified with BCA assay kit (Thermo Fisher Scientific). Thirty micrograms of protein was loaded, electrophoresed, and transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). Membranes were then blocked in 5% bovine serum albumin (BSA) solution for 1 h. After wash, membranes were incubated with primary antibodies against E-cadherin (1:1000, Abcam), N-cadherin (1:1000, Abcam), Snail (1:500, Abcam), c-Myc (1:500, Abcam), Cyclin D1 (1:2000, Abcam), Survivin (1:1000, Abcam), MET (1:1000, Abcam), phosphorylated MET (1:1000, Abcam), eIF4G1 (1:500, Abcam), phosphorylated eIF4G1 (1:500, Abcam) and GAPDH (1:4000, Abcam) at 4 °C overnight respectively. On the second day, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, which was visualized using enhanced chemiluminescence (ECL) substrates (Bio-Rad). Band intensity was quantified with ImageJ software. GAPDH was used as a normalization control.

Antibodies of PHGDH, SHMT1, eIF4A1, eIF4G1 and eIF4E were purchased from Abcam; antibodies of ZO-1 and E-cadherin were purchased from Cell Signaling Technology. ¹³C₆ glucose was purchased from Sigma-Aldrich (St. Louis, MO). Inhibitor CBR5884 was purchased from TOCIRS; inhibitor 4EGI-1 was purchased from Selleck; cisplatin and paclitaxel were purchased from J&K.