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. 2016 Apr 26;6:25133. doi: 10.1038/srep25133

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(a) Upper panel: Coomassie blue stained image of purified wildtype (GST-X) and LL-EE mutant (GST-D11) X-domain. Lower panel: Western blot image of upper panel samples, probed using anti-X antibody. (b) Left panel: Coomassie blue stained image of purified ORF3-His protein. Right panel: Western blot image of left panel sample, probed using anti-His antibody. (c) Left panel: Coomassie blue stained image of purified methyltransferase-His (Met-His) protein. Right panel: Western blot image of left panel sample, probed using anti-His antibody. (d) Pull down of His-tagged ORF3 protein using wild type (GST-X) or LL-EE mutant (GST-D11) X-domains as bait, revealed by immunoblotting using anti-His antibody (upper panel) and anti-X- antibody (lower panel). Only GST was used to control specificity of the binding (Lane 4). 40% of ORF3-His (10 ng) used in the pull down assay was loaded as input (Lane1). (e) Pull down of His-tagged methyltransferase using wild type (GST-X) or LL-EE mutant (GST-D11) X-domains as bait, revealed by immunoblotting using anti-His antibody (upper panel) and anti-X- antibody (lower panel). Only GST was used to control specificity of the binding (Lane 4). 40% of methyltransferase-His (10 ng) used in the pull down assay was loaded as input (Lane1). (f) Pull down of His-tagged ORF3 and His-tagged methyltransferase proteins using GST-X protein as bait, revealed by immunoblotting using anti-His-antibody (upper and middle panels) and anti-X antibody (lower panel). 50% of methyltransferase-His (10 ng) and ORF3-His (4 ng) used in the pull down assay was loaded as inputs in Lane 9 (upper panel) and lane 10 (middle panel), respectively. Relative band intensities of upper and middle panel images are plotted in the graph. (g) Quantitative α-galactosidase assay of Y2H gold transformants represented in Table 3. Normalized α-galactosidase units are plotted as ± SEM of triplicate samples.