Fig. 3.

NS5 interacts with p53. A and B The p53 protein was immunoprecipitated by NS5. HEK293T cells, transfected with plasmid encoding NS5-FLAG or vector, were lysed and immunoprecipitated with anti-FLAG beads. The elution from anti-FLAG beads were separated by SDS-PAGE and stained by sliver-staining (A). The whole lanes of NS5 or vector were analyzed by mass spectrometry (MS) assay and p53 were detected (B). C and D NS5 and p53 was immunoprecipitated by each other. HEK293T cells were co-transfected with the indicated plasmids and then co-immunoprecipitation assay was performed with anti-FLAG beads. The endogenous p53 (C) and NS5-HA (D) were detected by Western-blot. E The ZIKV-encoded NS5 was immunoprecipitated by p53. hNPCs infected by ZIKV were lysed and immunoprecipitated with anti-p53 antibody. The NS5 protein was detected by Western-blot. F NS5 and p53 co-distributed in sucrose gradient centrifugation experiment. hNPCs and HEK293T cells that expressed NS5 were lysed and fractionated on 10%–40% (v/v) of sucrose gradients and detected by Western-blot. G NS5 interacted with p53 in vitro with GST pull-down assay. The p53-GST and GST proteins were expressed in Escherichia coli BL21(D3) cells and purified to incubate with NS5-expressing HEK293T cells lysate, and then bound with GST beads. The protein interactions were detected by Western-blot.