Figure 4

The NCAM binding to PHE-CoV S protein. Lane 1-3, NCAM involves in recognition by PHE-CoV S protein. Supernatants of 293T cells transfected with plasmid encoding soluble NCAM. The 293T cells were added fusion S protein (6 mg) and incubate for 2 h at 4°C. The cells were lysed in 500 μl of radioimmune precipitation buffer. The 10-ml aliquot of lysate was incubated with 300 μl of glutathione-Sepharose beads conjugated with fusion anti-S protein antibody and gently rocking on a orbital shaker overnight at 4°C. The sepharose beads are boiled for 5 min to dissociate the immunocomplexes from the beads. The supernatant was electrophoresed through 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were blocked at room temperature for 3 h with 3% BSA in PBS containing Tween 20 (0.05%) and then incubated overnight with the anti-NCAM protein antibody. The proteins was analyzed by western blotting. Lane 4-6, Sf3b2, Hdac2 and RPS13 were not immunoprecipitated with S protein. Lane 7, 293T cells transfected with vector alone were negative controls.