Ecl reagent supersignal west pico chemiluminescent substrate

TRI^® reagent was purchased from Sigma Aldrich; High-Capacity cDNA Reverse Transcription Kit, Power SYBR Green Supermix and TaqMan^® Gene Expression Master Mix from Applied Biosystems; antibodies were purchased from Santa Cruz Biotechnology, USA; ECL reagent (SuperSignal^® West Pico Chemiluminescent Substrate) was procured from Thermo Scientific, USA; rabbit anti-cMyc antibody was purchased from Cell Signaling Technology; tissue culture media, serum and additional reagents were procured from HIMEDIA; RNase and cloning kits were purchased from Fermentas; HiPerfect transfection and silencing reagents and QIAprep^® Spin Miniprep kit were procured from Qiagen. Restriction enzymes were purchased from Thermo Scientific, USA.

Tissues were cut and digested by RIPA buffer (Beyotime Biotechnology, Shanghai, China) and the concentration of proteins were measured using BCA™ Protein assay kit (ThermoFisher Scientific). Total 30 μg protein was separated by SDS PAGE gels and transferred to 0.2 µm PVDF membrane (Millipore). After blocking with 5% fat free milk, membrane was incubated with primary antibodies (anti-GFPT2, Thermo Fisher Scientific, PA5-26290, 1:1,000 dilution; anti-GAPDH, abcam, ab8245, 1:5,000 dilution) at 4°C overnight. The next day, the membrane was incubated with secondary antibodies (Cell Signaling Technology, #14708 or #14709, 1:10,000 dilution) for 2 hrs at room temperature. Then the membrane was exposed after using ECL reagent Supersignal West Pico chemiluminescent Substrate (ThermoFisher Scientific).