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monomeric EGFP were used as a brightness standard and were imaged under the
same conditions as EGFP-VP40 and respective mutations (see Fig. 19.1 ).
The brightness of the EGFP was used as the brightness of the monomer as de-
scribed previously ( Adu-Gyamfi et al., 2012 ) where the average brightness of a
monomer was 0.104. This allowed for selection of oligomeric size based upon mul-
tiple of the monomer (see Figs. 19.1 and 19.2 ). Thus, the selection window for each
species is based upon the average brightness, which will yield an average population
of each species in the respective area of analysis (see Figs. 19.1 and 19.2 ).
FIGURE 19.1
Brightness analysis of VP40 in HEK293 cells. (A) Membrane protrusion sites emanating from
the PM were inspected with total internal reflection microscopy (TIRF) microscopy.
(B) TIRF average intensity image of a HEK293 cell transfected with plasmid expressing EGFP
demonstrates sites of signal enrichment and a number of sites of membrane protrusions
and viral egress. (C) Brightness image (variance/intensity) of the same cell demonstrates the
enriched sites of VLP egress where significant EGFP signal (red) is detected.
(D) Brightness versus intensity plot displaying monomers (brightness of 1.104) (red box),
dimers (blue box), and hexamers (green box). (E) Brightness distribution of VP40 with
selected pixels from D displaying localization of monomers (red), dimers (blue), and
hexamers (green). (F) Frequency versus apparent brightness plot demonstrates the extensive
oligomerization of VP40 at or near the PM of HEK293 cells. The apparent brightness of a
monomer is 1.104 indicating the significant frequency of a monomer but extensive enrichment
of oligomers up to an apparent brightness of 12. Scale bar
m.
This research was originally published in Adu-Gyamfi et al. (2013) .
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