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. Author manuscript; available in PMC: 2012 Dec 5.
Published in final edited form as: Virology. 2011 Oct 7;421(1):51–60. doi: 10.1016/j.virol.2011.08.008

Figure 4. Analysis of Nck-PKR interactions in WNV-infected cells.

Figure 4

(A) Analysis of colocalization of PKR with Nck. BHK cells were mock-infected or infected with WNV Eg101(MOI of 5). At 30 h after infection, cells were permeabilized, fixed and blocked overnight. Cells were stained with mouse monoclonal anti-PKR and rabbit polyclonal anti-Nck antibodies and then with AlexaFluor488 (green) and AlexaFluor594/555 (red) conjugated secondary antibodies. (B) Analysis of Nck protein levels in WNV-infected BHK cells. Cells were mock-infected (M), or infected with WNV Eg101 (MOI of 5) for the indicated times. NS3, Nck-1, and actin were detected by Western blotting after separation of proteins by 10% SDS-PAGE. (C) Co-immunoprecipitation of Nck and PKR. BHK cells were mock-infected (M) or infected with WNV Eg101 (MOI of 5). At 24 h after infection, cells were lysed and S2 fractions were prepared. Rabbit anti-PKR antibody or a rabbit anti-Nck antibody was used for immunoprecipitation; rabbit IgG was used as a control antibody.. Immunoprecipitated proteins were separated by 10% SDS-PAGE and detected by Western blotting using a rabbit anti-Nck antibody (top panel) or a mouse anti-PKR antibody (bottom panel). (D) Analysis of PKR phosphorylation in WNV-infected Nck-knockout cells. Nck-1,2−/− MEFs were mock-infected (M) or infected with WNV Eg101 (MOI of 5) for the indicated times or treated with 100 U/ml universal type I IFN for 24 h (IFN). NS3, total PKR, phopho-Thr451 PKR, total eIF2a and phospho-Ser51 eIF2a were detected by Western blotting after separation of proteins by 10% SDS-PAGE. Blots shown are representative of at least two independent experiments.