4.5. GST Pulldown Assay
For expression of the GST-tagged NS5A protein, the NS5A gene was subcloned into the pGEX-6p-1 vector (catalog no. 28-9546-48; GE Healthcare, Piscataway, NJ, USA), creating pGEX-NS5A. The GST or GST-NS5A fusion protein expressed in E. coli BL21(DE3) were purified with the glutathione-Sepharose 4B resin (catalog no. 10049253; GE Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. Briefly, the expression of GST or GST-NS5A protein was induced by addition of 1 mM isopropylthiogalactoside (IPTG) (catalog no. ST098; Beyotime, Shanghai, China).The bacterial cells were harvested and resuspended in cold phosphate-buffered saline (PBS) containing 1 mg/mL protease inhibitor cocktail (catalog no. 11873580001; Roche, Penzberg, Germany), followed by mild sonication. Insoluble components were removed by centrifugation at 10,000 ×g for 20 min. Subsequently, the soluble GST or GST-NS5A protein was incubated with the glutathione-Sepharose 4B resin for 4 h at 4 °C. The resins were washed four times with cold PBS and incubated with 300 μL of the lysate of the HEK293T cells transfected with pMyc-eEF1A for 2 h at 4 °C. After an extensive wash with PBS, the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting using the anti-Myc MAb (1:1000) (catalog no. M4439; Sigma-Aldrich) and an anti-GST polyclonal antibody (PAb) (1:2000) (catalog no. AB101; Tiangen, Beijing, China).