Figure 4. Nef:MHC-I interaction occurs within Rab7-positive late endosomes.

(a) HeLa cells were co-transfected with Nef-V_C and MHC-I-V_N-Flag encoding constructs along with constructs encoding wildtype mCherry-Rab7 or dominant negative mCherry-Rab7 (mCherry-Rab7-DN). BiFC fluorescence (green) was visualized under the FITC channel, mCherry-Rab7 (red) was detected under the Cy3 channel. Nuclei were stained with DAPI (scale bars represent 10 μm). (b) Co-localization was quantified by using the Pearson’s correlation through the JaCoP Plug-in on ImageJ. Error bars were calculated by quantification of at least 40 cells between 3 independent experiments. Western blot analysis for mCherry to confirm expression levels of Rab7 and Rab7-DN, with actin as a loading control. (c) Cells were transfected with Nef-V_C and MHC-I-V_N-Flag and immunostained for Rab7 and subsequently imaged utilizing ground state depletion microscopy (GSDM). (d) A histogram plotting the intermolecular distances between the nearest neighbor, representing either BiFC:Rab7 (Solid line) or BiFC:Simulated random positions (Sim. Positions; Dashed line). (e) A graphical representation of the fraction of co-localized particles observed in (d) which were observed to be under the cut-off value of ~40 nm. Error bars were calculated by quantification at least 10 cells in 3 independent experiments (**indicates p-value < 0.01, ***indicates p-value < 0.001).