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Chen
et al. Vet Res (2021) 52:73
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.