Fig. 4.

The NS2–CHD3 interaction played a significant impact on the distribution of Crm1 and NS2. a COS-1 cells were fractionated and cell fractionation was confirmed with antibodies against the following subcellular marker proteins: tubulin (cytoplasmic, cyt), HMGB1 (mainly nucleoplasmic, nuc), TFIIB (low-salt chromatin fraction, ch150) and histone H3 (whole chromatin fraction). b The distribution of endogenous CHD3 during WD infection. Cells were infected with WD virus (MOI 0.1). At 0 h and 18 h p.i., the cells were harvested and analyzed via western blotting. c The effect of WD infection (MOI 3) on the location of NP and NS2 at early stage of the infection (4 h p.i.). d a Knockdown of CHD3 using siRNA resulted in the diffusion of NS2 into all subcellular fractions and less Crm1 in the >ch500 fraction. COS-1 cells were transfected with si-CHD3 or si-NC; then, 24 h later the cells were infected with WD virus (MOI 3) for 4 h. The relative quantities of the NS2 (b) and Crm1 (c) in each fraction were analyzed using the software Image J (NIH) (*p < 0.05, **p < 0.01, n = 3). e NLS–cCHD3 changed the Crm1 and NS2 distribution under WD infection. COS-1cells were transfected with NLS–cCHD3 or empty vector as a control; 24 h later, the cells were infected with WD virus (MOI 0.3) for 10 h. f IFA assaying the effect of NLS–cCHD3 overexpression on location of NS2 and Crm1 at early stage of infection. COS-1 cells transfected with plasmid expressing NLS-Myc-cCHD3 (top) or untransfected (bottom) were infected with WD-Flag-NS2 virus. The location of NLS-Myc-cCHD3 and NS2 was marked by IFA. Colocalization was analyzed using Image J and the coefficients confirmed the strong colocalization. GAPDH was used to control the protein loading