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. 2013 Nov 15;8(11):e80617. doi: 10.1371/journal.pone.0080617

Figure 5. Propeptide influences Cul3(wt) association and nucleolar localization of the pVII(wt) protein.

Figure 5

(A) The propeptide 1-24 sequence enhances Flag-Cul3(wt) interaction with the pVII protein. GST, GST-pVII(wt), GST-pVII(Δ24) and GST-pVII(K26R/K27R) were used in a GST-pull down assay with HEK293T whole cell lysate expressing Flag-Cul3(wt) protein. Filled arrow indicates the migration of the full-length pVII(wt), pVII(Δ24) and pVII(K26R/K27R) proteins. Non-filled arrow indicates migration of the GST protein. Asterisks (*) indicate the presence of truncated versions of the GST-pVII proteins. Hyphen (-) indicates an empty lane on the SDS-PAGE. (B) 1-24 propeptide interacts with the Flag-Cul3(wt) protein. Peptide pull-down assay with the synthetic 1-24 propeptide (lane 3) and control peptide (lane 4) was performed with HEK293T cell lysates expressing Flag-Cul3(wt) protein. Non-peptide coupled magnetic beads were also used as the specificity control (lane 2). Input represents 10% of the HEK293T cell lysates expressing Flag-Cul3(wt) protein. (C) Localization of the pVII(wt)Flag, pVII(Δ24)Flag and pVII(K26R/K27R)Flag proteins in the nucleus. HeLa cells were transfected with the respective plasmids for 24 hours and were subjected to indirect immunofluorescence analysis. The Flag-tagged pVII proteins and fibrillarin were simultaneously stained and visualized. Nuclei were counterstained with DAPI. Quantification of Flag-tagged pVII protein localization in HeLa cells (n=40) is shown on the right side of the Figure 5C.