Figure 4.

IMPDH2 associates with Nsp14 and is required for IL-8 upregulation by Nsp14. (A) HEK293T cells were transiently transfected with the vector expressing FLAG-Nsp14 or V5-IMPDH2, alone or together. Cell lysates were prepared and subjected to protein co-immunoprecipitation (co-IP) assays using anti-FLAG or control IgG antibody. Precipitated protein samples were analyzed by protein immunoblotting using anti-V5 and anti-FLAG antibodies. (B) HEK293T cells were transiently transfected with the empty vector (V) or FLAG-V5-Nsp14 vector. Cell lysates were prepared and subjected to protein co-IP assays using an anti-FLAG antibody. Precipitated protein samples were analyzed by protein immunoblotting using anti-V5 and anti-IMPDH2 antibodies. (C) HEK293T cells were transiently transfected with IMPDH2 or non-targeting (NT) siRNAs. The mRNA level of IMPDH2 was measured and normalized to siNT. (D) HEK293T cells transfected with IMPDH2 or NT siRNAs were further transfected with V5-FLAG-Nsp14 or empty vector. These cells were untreated or treated with TNF-α. Total RNAs were extracted. IL-8 mRNA was analyzed and normalized to the siNT and empty vector-transfected group. Results were calculated from 3 independent experiments and presented as mean +/- standard error of the mean (SEM). (*p<0.05; ** p<0.01; **** p<0.0001; by unpaired Student’s t-test).