Fig. 3. Intermixing of both small- and large-scale clusters of MHC-I and MHC-II proteins in the plasma membrane of JY cells points to their dynamic nature. Two samples of JY cells were separately labeled with antibodies tagged with different fluorescent labels. (For epitopes labeled by the antibodies see text below.) These samples were subsequently fused with each other using PEG. Cells were then incubated at 37°C in all the cases except where it is indicated. The FRET efficiency characterizing small-scale clustering of proteins was determined using the photobleacheing FRET approach 0 and 80 minutes after cell fusion (white and black columns, respectively, in A). Overlap of large-scale clusters was determined in a SNOM (B). When the two proteins labeled by the antibodies were different, overlapping cluster areas were normalized by dividing the double-colored area with the area of either of the protein clusters. Because the procedures yielded the same results within experimental error, only the normalization with the area of the first protein cluster is presented. As positive controls samples were labeled with antibodies against both proteins (left-hatched columns), and FRET efficiency and overlap percentage was determined similarly. Results are mean±s.e.m. Cell-surface receptor pairs were as follows: (A) MHC-I and MHC-I, (B) MHC-I and MHC-I, incubation on ice, (C) MHC-I and MHC-I in the presence of AlF₃, (D) MHC-II and MHC-II, (E) MHC-I and MHC-II.