Figure 2

Screening and identification of NS1 mRNA interacting host proteins. A Experimental design for pulldown assays and identification of NS1 mRNA associated cellular proteins. NS1 mRNA and LacZ mRNA were biotinylated by transcription in vitro, and incubated with PK-15 total cell lysates. B Coomassie bright blue staining of biotinylated NS1 mRNA associated proteins. C The gene ontology (GO) analysis on NS1 mRNA interacting with host proteins. The GO analysis was performed by mRNA processing of the biological process on NS1 mRNA interacting with host proteins using webgestalt. D HEK293 cells were transfected with different expression vectors containing Flag tagged host proteins for 24 h. RNA-pulldown was performed with streptavidin beads, followed by western blotting using the anti-Flag antibodies. E Western blotting identified the interaction between NS1 mRNA and endogenous SYNCRIP protein in PPV-infected PK-15 cells by RNA-pulldown. F RNA-immunoprecipitation identified the interaction between NS1 mRNA and endogenous SYNCRIP protein in PPV-infected PK-15 cells. An anti-SYNCRIP antibody or negative control IgG was used to pull down RNA–protein complexes. Recovered cDNA from PPV-infected PK-15 cells was examined for viral RNA by PCR with primer sets of NS1 and GAPDH, Y-PPV plasmid was used as a template for positive controls of PCR. G Laser confocal identify the interaction between NS1 mRNA and endogenous SYNCRIP protein in PPV-infected PK-15 cells. H Identification of GST and GST-SYNCRIP protein purification. (I) EMSA identified the interaction between NS1 mRNA and endogenous SYNCRIP protein. Scale bar = 10 μm.