β3-AAA nAChRs and CI-976 treatment decrease interaction between α6 and COPI. NFRET measurements were made using α6-mCherry and εCOP-eGFP transfected in Neuro-2a cells (along with β2 and β3wt or β3AAA subunits). (A) Representative NFRET images between α6-mCherry and εCOP-eGFP in Neuro-2a cells. Bars, 10 µm. (B) Summed histograms of pixel-based NFRET measurements, representative of four independent NFRET assays (n = 20 cells). C–E show analysis of the same group of cells (n = 28 cells). (C) Another of the four experiments. Scatter-box plot for the mean NFRET values of individual cells. The larger black diamond (◆) in the box plot represents the cell-based mean of each condition. (D) α6-eGFP fluorescence in COPI vesicles for α6-eGFPβ2β3AAA nAChRs and α6-eGFPβ2β3WT nAChRs (without and with 20 µM CI-976). (E) Pearson correlation for the colocalization of α6-eGFP* fluorescence with εCOP-GFP fluorescence (n = 28 cells). *, P < 0.05; **, P < 0.01; ***, P < 0.005 (ANOVA).
