FIGURE 2: Homo-dimerization in eukaryotic membranes. (A) Schematic representation of BiFC Assay. TM-driven oligomerization results in complementation of two non-fluorescent halves (amino-terminus (VN) and carboxy-terminus (VC)) of the Venus Fluorescent Protein (VFP). (B) Mean relative fluorescence of chimera homo-oligomerization in HEK293T cells (GpA (VN-GpA/VC-GpA), 17L (VN-17L/VC-17L), 19L (VN-19L/VC-19L), 23L (VN-23L/VC-23L), 25L (VN-25L/VC-25L), 27L (VN-27L/VC-27L), 29L (VN-29L/VC-29L), H2 (VN-H2/VC-H2)). Error bars indicate standard deviation obtained from at least 4 independent replicates (H2 was used as a negative control, **** < 0.0001). A color intensity code is used to highlight dimerizing (green) and non-dimerizing (white) transmembrane segments, while positive dimerization control (GpA) is shown in black. (C) Western Blot show chimera’s expression levels detected by α-c-Myc antibody. (D) The contribution of Gly for the dimerization of TM chimeras. Relative fluorescence of chimera homo-oligomerization in human-derived HEK293T cells (GpA [VN-GpA/VC-GpA (depicted in black)], H2 [VN-H2/VC-H2] (white), 17L [VN-17L/VC-17L], 17L_I [VN-17L_I/VC-17L_I], 29L [VN-29L/VC-29L], 29L_I [VN-29L_I/VC-29L_I). The bars represent mean values of chimera homo-oligomerization, and error bars denote standard deviation obtained from 3 independent experiments (p-values of Student’s t-test: * < 0.05, ** < 0.01, ns (non-significant)). Color intensity is used to highlight dimerizing (significantly different from H2 control, green) and non-dimerizing (white). Light green is used to indicate those samples (where the Gly residues of the dimerization domain have been substituted with Ile) which fluorescence values are significantly higher than the H2 control and at the same time lower than the corresponding non-mutated control. (E) Cellular fractionation of chimera expressing eukaryotic cells. Subcellular fractionation of HEK293T cells expressing BiFC chimeras (c-Myc tagged) (VN-23L, VN-25L and VN-GpA) or EYFP (Flag-tagged) (used as a soluble marker). Soluble fraction (Sol) and Membrane fraction (MB).