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. 2014 Feb 19;9(2):e86745. doi: 10.1371/journal.pone.0086745

Figure 10. WEEV nsP3 interaction with host IKKβ.

Figure 10

A) U87MGs were transfected in a 6-well plate with 5 µg of pUC19 and WEEV_nsP3_HA for 24 hours. Cell lysates were resolved using SDS-PAGE and subsequently immunoblotted with V5 antibody and β-actin served as a loading control. B) U87MGs were transfected with WEEV_nsP3_V5; cells were fixed after 24 hours and stained with antibodies against the endogenous IKKβ and the V5 tag. Cells were incubated with appropriate secondary Alexa Fluor antibodies and the nuclei stained with DAPI. Co-localization of IKKβ with WEEV_nsP3_V5 (yellow) was observed as shown by the arrows. B) Panels E–H serve as an example of transfected cells in a given field of view that show co-localization of IKKβ and WEEV_nsP3_V5 24 hours post transfection. Panels I-L represent magnified images of other cells showing co-localization of IKKβ and WEEV_nsP3_V5. Panel M is a magnified image of panel L. The co-localization was confirmed by Z-stack analysis. Co-localization was calculated to be approximately in 61% of cells (163 cells were counted of which 44% demonstrated expression of nsP3. Of those cells that expressed nsP3, 61% showed co-localization of both proteins). Images were taken using Nikon Eclipse TE2000-U at 60× magnification and are representative of 2 independent experiments.