NLS-mediated interplay between C protein and importin-α. (A) pWNLS transfected into Vero
Ipragliflozin shows intense green fluorescence in the nucleoli region (arrows, i) in contrast to pTCSΔ39-transfected cells where only cytoplasmic fluorescence is observed even after 24 h post-transfection (arrows, ii). (B) Co-immunoprecipitation. Vero cells transfected with RNAs in vitro transcribed from pWNV/pWNVΔNLS were immunoprecipitated with anti-importin-α Ab and immunoblotted with anti-C Ab. The presence of band in Lane 2 and absence of band in Lane 3 confirm that C protein–importin-α binding is mediated by the NLS motif of C protein. (ii–v) Precipitation control, input controls and mouse isotype Ab controls. (C) Co-immunoprecipitation. Vero
cells were transfected with RNA transcribed from pWNS/pWNSM1/pWNSM2/pWNSM1M2/pWNS4243/pWNSΔNLS plasmids. At 24 h post-transfection, immunoprecipitation was performed using anti-importin-α (i) or anti-importin-β (ii) Ab followed by immunoblotting using anti-WNVC Ab. The presence of bands in Lanes 2–6 and absence of bands in Lane 7 in anti-importin-α–β antibody-immunoprecipitated samples confirm the binding between importin-α/β and mutated C proteins except for pWNSΔNLS. (iii–v) Input controls.
