Co-immunoprecipitation (co-IP) Assays

Plasmid pcDNA-NS4B-Flag was transfected alone or co-transfected with pcDNA-Rab5-Myc into PK-15 cells. Plasmids pcDNA-NS5A-Flag and pcDNA-Rab5-Myc as well as pcDNA-NS4B-Flag and pcDNA-Rab2-Myc were co-transfected in to PK-15 cells and served as negative control. After 48 h post transfection, the cells were resuspended in western blot and IP lysis buffer with PMSF, and the lysate was used for co-IP assays utilizing ANTI-FLAG M2 Affinity Gel or Anti-c-Myc Agarose Affinity Gel antibody according to the manufacturers’ protocol. Briefly, resin (50 μL) was centrifuged at 8,000 g for 30 s to remove the stock solution and washed twice with TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Then, 1 mL of cell lysate was added to the rinsed resin and rocked gently overnight at 4°C. Thereafter, the resin was washed three times with TBS, and the agarose beads pellet was resuspended in 2 × SDS loading buffer followed by boiling for 10 min. Finally, SDS-PAGE and immunoblotting were conducted to detect the supernatant.