4.6. Coimmunoprecipitation Assay
For Co-IP assay, the p3×Flag-NS5A and pMyc-eEF1A were cotransfected into HEK293T cells as described above. The cells were collected at 48 hpt, washed two times with cold PBS, and lyzed with the CHAPS lysis buffer (1% CHAPS, 150 mM NaCl, 5 mM MgCl2 and 10 mM Tris-HCl [pH 7.5]) containing 1 mM PMSF (catalog no. ST506; Beyotime) and 1 mg/mL protease inhibitor cocktail at 4 °C for 1 h. The cell lysate was centrifuged at 13,000 ×g for 20 min at 4 °C. The clarified lysate was treated with a mixture of RNase A (25 U/mL) (catalog no. 2158; TaKaRa, Dalian, China) and RNase T1 (1 U/µL) (catalog no. PF0059; Fermentas, Burlington, Canada) for 1 h at room temperature prior to Co-IP analysis. The lysate was precleared with the Protein G-Agarose (catalog no. 11243233001; Roche) at 4 °C for 4 h followed by incubation with the indicated antibodies at 4 °C for 4 h, and the Protein G-Agarose was then added and incubated at 4 °C for 2 h. The Protein G-Agarose was washed three times with PBS, and the bound proteins were separated by SDS-PAGE followed by Western blotting using the indicated antibodies.