Fig. 2.

Viroplasmic localization of NSP2 and association with NSP5 is disrupted in CK1α-silenced cells. MA104 cells were transfected with a siRNA pool targeting CK1α 48–72 h before RV infection. RV-infected cells (MOI 5) were fixed at 6 hpi before (A) dNSP2 or (B) vNSP2 (green) and NSP5 (red) were detected using monoclonal antibodies or polyclonal antisera, respectively. Cell nuclei were stained with DAPI (blue). Images were obtained by confocal microscopy. (Scale bar: 10 µm.) (C) WB to detect NSP2. Equivalent aliquots of RV-infected cell lysate were loaded onto nonreducing and reducing gels and NSP2 was detected with either anti-dNSP2 (nonreducing gel) or anti-vNSP2 (nonreducing gel) antibodies. Anti-tubulin (reducing gel) was used as a loading control. The CK1α-silenced lysate was run on the same gel as the controls; however, intervening lanes were removed. (D) WBs for NSP5 and CK1α. Cell lysates were separated under reducing conditions to detect NSP5, CK1α, and tubulin. To visualize the hypophosphorylated bands of NSP5, the Middle panel was loaded with less lysate than the Upper panel. An irrelevant (Irr.) siRNA was used as a control. MW, apparent molecular weight.