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. 2014 Apr 15;15(6):615–626. doi: 10.1111/mpp.12120

Figure 2.

figure

Interaction between Turnip mosaic virus (TuMV) VPg (viral protein genome‐linked) and Chinese cabbage eIF(iso)4E [eukaryotic initiation factor(iso)4E]. Bait plasmid pEG202 was used to express a TuMV VPg fusion protein, and prey plasmid pJG4‐5 was used to express wild‐type and mutated eIF(iso)4E proteins. (a) Yeast transformants were streaked on minimal medium agar plates containing 40 μg/mL X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside) to assay the expression of the lacZ reporter gene, indicated by the development of a blue colour. (b) Quantitative liquid culture assay using chlorophenolred‐β‐d‐galactopyranoside (CPRG) as substrate was employed to calculate β‐galactosidase activity. One unit of β‐galactosidase equals the amount that hydrolyses 1 μmol of CPRG to chlorophenol red and d‐galactose per minute per cell. Neg, negative control; WT, susceptible WT Samjin eIF(iso)4E. Different letters denote statistically significantly different values [analysis of variance (ANOVA), P ≥ 0.05]. (c) Immunoblot of co‐immunoprecipitation assays between eIF(iso)4E (WT and mutants) and TuMV VPg. Immunoprecipitation (IP) was carried out with anti‐haemagglutinin (HA) agarose beads, and immunoblotting (IB) was performed with the indicated antibodies. Green fluorescent proteins (GFPs) (HA‐GFP and FLAG‐GFP) were used as negative controls.