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. 2008 Jul 4;4(7):e1000096. doi: 10.1371/journal.ppat.1000096

Figure 5. E3 interacts with ISG15 protein.

Figure 5

A. PKR+/+ MEFs were treated with mouse IFN-α (100 units/ml) during 10 hrs and then infected with 3 PFU/cell of WR, or MVA or VVΔE3L or VVE3LΔ83N or VVE3LΔ26C for 16 hours. Cell extracts were collected at 16 hpi and immunoprecipitated using anti-ISG15 serum, thoroughly washed and immunocomplexes analysed by SDS-PAGE and subjected to immunoblotting with antiserum to E3 or ISG15. B. PKR+/+ cells were treated as above and cell extracts were collected at 16 hpi and immunoprecipitated using anti-E3 (with or without RNase treatment (10 µg for 15 min at room temperature); or without antibody; or using a pre-immune serum, thoroughly washed and immunocomplexes analysed by SDS-PAGE and subjected to immunoblotting with antiserum to E3 or ISG15. C. PKR+/+ MEFs treated as in A were fixed at 16 h.p.i. and processed for immunofluorescence analysis by confocal microscopy using antibodies directed against ISG15 (red), E3 (green), and TOPRO for staining nuclei (blue). Merged images are presented in the lower panels. Cells were visualized by confocal immunofluorescence microscopy. D. Immunoprecipitation and immunoflurescence of PKR−/− MEFs was performed as in A or in C.