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. 2011 Dec;85(24):12939–12949. doi: 10.1128/JVI.05177-11

Fig. 2.

Fig. 2.

Interaction of PCBP1 and PCBP2 with PRRSV nsp1β. Co-IP of HA-PCBP1 (A) and HA-PCBP2 (B) with Flag-nsp1β. Indicated plasmids (shown on top) were transfected into 293T cells, and at 48 hpt, whole-cell lysates (WCL) were immunoprecipitated with anti-Flag or anti-IgG. After SDS-PAGE separation, proteins were detected by immunoblotting with antibodies indicated on the left. A 5% aliquot of WCL was also probed similarly to confirm the expression of the proteins. The identities of various protein bands are indicated on the right. (C) GST pulldown assay. Glutathione beads conjugated to GST or GST-nsp1β fusion protein were incubated with recombinant PCBP1 (left) or PCBP2 (right). After washing, proteins were eluted from the beads and SDS-PAGE was performed. The presence of PCBP1 and PCBP2 was detected by anti-HA antibody. GST and GST-nsp1β expression was confirmed by use of anti-GST antibody. (D) Co-IP of PRRSV nsp1β with endogenous PCBP1. PRRSV-infected (+) or mock-infected (−) MARC-145 cells were used for IP with anti-PCBP1 antibody and immunoblotted with antibodies shown on the left. The identities of various bands are shown on the right. (E) Colocalization of nsp1β with HA-PCBP1 and HA-PCBP2 by confocal microscopy. MARC-145 cells were mock infected or infected with PRRSV (the VR-2332 strain was used here, since the nsp1β monoclonal antibody is against this virus) for 6 h before being transfected with the PCBP protein-expressing plasmids. Cells were fixed 18 hpt and subjected to indirect immunofluorescence to detect nsp1β (green) and HA-PCBP1 or PCBP2 (red) by using mouse anti-nsp1β and rabbit anti-HA antibodies. Position of nucleus is indicated by DAPI (4′,6-diamidino-2-phenylindole) (blue) by staining in the merge image (far right). (F) Effect of RNase treatment on PCBP1/2-nsp1β interaction. Extracts of 293T cells overexpressing the different proteins were treated or untreated with RNase A and T1 for 1 h before IP and immunoblotted with antibodies shown on the left. Ethidium bromide-stained agarose gels show total RNA present with or without RNase treatment. WB, Western blotting.