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. 2017 Jul 4;9(7):169. doi: 10.3390/v9070169

Figure 5.

Figure 5

The 2BC non-structural protein of EV-A71 interacts with syntaxin-17 (STX17) and synaptosome-associated protein of 29 kDa (SNAP29). (A) Yeast two-hybrid assays of 2BC and STX17. Yeasts were transformed with pGBKT7 or pGBKT7-STX17 together with pACT2 or pACT2–2BC. Yeasts were then grown on selective medium without tryptophan (-W) or without leucine, tryptophan, and histidine (-LWH); (B) HeLa cells were transfected with pDEST27 or pDEST27–2BC in combination with pCherry-STX17 for 48 h. The transfected cells were examined under a confocal microscope with objective 63× (scale bar 10µm) for the presence of co-localization; and (C) the Pearson’s correlation coefficient was determined; (D) HEK-293T cells were co-transfected with FLAG-STX17 and GST-2BC for 48 h. Lysates were collected for co-immunoprecipitation (IP) using glutathione (GST) sepharose. Western Blot was performed to detect FLAG-STX17 and GST fusion proteins; (E) RD cells were infected with EV-A71 for 8 hpi and lysates of infected cells were then harvested prior to co-immunoprecipitation (co-IP) assay using anti-STX17 and anti-SNAP29. The final IP eluates and discarded IP flow through were collected for WB to detect 2BC (anti-2C polyclonal antibody) and structural proteins (mAB979) of EV-A71. One-way ANOVA: * p < 0.05.