Figure 1. Bimolecular fluorescence complementation is observed between Nef and MHC-I.

(a) Left: Schematic representation of the BiFC reporter system. Right: Nef-V_C and either wildtype MHC-I-V_N-Flag or the indicated mutants were transfected into HeLa cells, 24 hrs later cells were fixed, and BiFC fluorescence (green) was observed under the FITC channel. Scale bars represent 20 μm. (b) Fluorescence intensities of Nef and MHC-I-V_N-Flag positive cells were quantified in ImageJ, minus the background signal, to observe a decrease in fluorescence in the presence of the MHC-I mutations (n = 100, *indicates p-value < 0.05, **indicates p-value < 0.01). (c) Flag and GFP specific Western blots were conducted to ensure equal expression of both the MHC-I-V_N-Flag mutants and Nef-V_C, respectively. An actin specific Western blot was conducted as a loading control.