Effect of α4 and β2 ER export motifs, β2 ER retention/retrieval motifs, and nicotine exposure on ERES. (A) Representative confocal images of a Neuro-2a cell transfected with α4-eGFPβ2wt nAChRs and an ERES marker, Sec24D-mcherry. ROI representing Sec24D-mcherry ERES structures are demarcated, and a merged image of α4-eGFPβ2wt plus Sec24D-mcherry is shown. Bars, 5 µm. (B) Average number of ERES per cell for α4-eGFPL358Aβ2–, α4-eGFPβ2wt–, α4-eGFPβ2L349M–, α4-eGFPβ4wt–, and α4-eGFPL358Aβ4wt–transfected cells. (C) Scatter plot of number of ERES per cell for α4-eGFPL358Aβ2–, α4-eGFPβ2wt–, α4-eGFPL358Aβ4wt–, α4-eGFPβ4wt–, and α4-eGFPβ2L349M–transfected cells. (D) Number of ERES per cell for α4-eGFPβ2wt– and α4-eGFPβ2-365AAQA368–transfected cells. (E) Number of ERES per cell in untreated and nicotine-treated (0.1 µM for 48 h) cells transfected either with no additional proteins, with α4-eGFPβ2wt nAChRs, or with GluClαwtGluClβ-eGFP. The number of cells imaged is indicated in parentheses; data are mean values ± SEM. NS, not significant; *, P = 0.06; **, P < 0.01; ***, P < 0.001, two-tailed t test. The break on the horizontal axis indicates sets of experiments that were performed on different batches of cells; therefore, the absolute numbers of ERES may not be directly comparable between batches.