Figure 4.

Brightness analysis of VP40 and EGFP in HEK293T cells. (A) TIRF intensity image of a HEK293T cell transfected with plasmid expressing EGFP. (B) Brightness image of the same cell shows the lack of EGFP clustering. (C) Brightness versus intensity map for the EGFP expressing cell with monomers highlighted by the red box. (D) Selected pixels from C displaying the localization of monomeric EGFP in red. (E) TIRF intensity image of a HEK293T cell expressing EGFP-VP40. (F) Brightness distribution of the VP40 expressing cell in E demonstrates enrichment of EGFP-VP40 clustering on the plasma membrane. Specifically, EGFP-VP40 is clustered at sites of membrane protrusion that can also be viewed in Fig. S3. (G) Brightness versus intensity map of VP40 showing monomers (red box or lower box), trimers (blue box or middle box), and octamers (green box or upper box). (H) Selected pixels from G showing monomers (red), trimers (blue), and octamers (green). The majority of VP40 localized to the plasma membrane is present in the monomeric form. (I) Brightness versus intensity plot displaying hexamers (red box or lower box) and octamers (blue box or upper box). (J) Brightness distribution of VP40 with selected pixels displaying hexamers (red) and octamers (blue), which are significantly enriched at sites of membrane protrusions. (K) Brightness versus intensity plot displaying 16 mers (red box). (L) Image of the cell showing pixels with brightness of 16 mers (red) localized at sites of membrane protrusion. White scale bar = 18 μm on all panels.