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. 2012 Oct 16;4(10):2137–2161. doi: 10.3390/v4102137

Figure 1.

Figure 1

LCMV-NP D471G single amino acid mutant is affected in self-association. (A) Co-IP: Human 293T cells were co-transfected with 2 μg of the pC- LCMV-NP wt, single (Q362R and D471G), or double (Q362R/D471G) amino acid mutants HA-tagged, together with 2 μg of pC-LCMV-NP wt FLAG-tagged expression plasmid. As controls, LCMV-NP tagged versions were expressed individually together with 2 μg of empty pC to keep constant the total amount of transfected DNA. At 48 hpt, cell lysates were prepared and analyzed for protein expression levels by WB using anti-HA or anti-FLAG polyclonal antibodies (i). GAPDH was used as a loading control. Cell lysates were immunoprecipitated with anti-FLAG affinity agarose beads (ii) and analyzed by WB with the indicated antibodies (left). Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to the signal of cells co-transfected with HA- and FLAG-tagged wt NPs, as described in Material and Methods. (B) M2H: Human 293T cells were co-transfected in triplicate with 2 μg of the indicated wt, single, or double amino acid mutant pC-VP16-tagged expression plasmids, together with 2 μg of LCMV-NP-GAL4 expression plasmid, along with 1 μg of the pG5 GFP/FFL dual reporter plasmid, and 0.1 μg of the pRL SV40 expression plasmid to normalize transfection efficiencies. At 72 hpt, GFP expression was assessed using fluorescence microscopy and cell extracts were prepared to determine the strength of NP-NP interaction using the Promega dual-luciferase reporter assay and a Lumicount luminometer. As negative control, we transfected cells with pC-NP-GAL4 together with pC-VP16. Representative fields of transfected cells are illustrated (i). Reporter gene activation (FFL) is shown as percentage (%) of LCMV NP-NP wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies with the Renilla luciferase expression plasmid pRL SV40 (ii). Cell lysates were used to detect expression of LCMV-NP wt and mutants by WB using an anti-VP16 polyclonal antibody (iii). GAPDH was used as a loading control. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP, as described in material and methods.