Subcellular localization of α4β2 and α4β4 nAChRs depends on the β subunit. (A) Representative TIRF images of Neuro-2a cells expressing a PM reporter (PM mcherry) or α4-mcherry plus β2wt along with either ER-Tracker Green or α4eGFP plus β2wt or α4eGFP plus β4wt. Each column is a different cell. Red and green fluorescence channels are separately shown in the top two rows, and merged images are in the bottom row. Panel labels indicate the fluorophore and transfected proteins. B1, pseudo-colored TIRF image of α4eGFP plus β2wt–transfected cell; B2, ER fluorescence signal from cell in B1 after subtraction of PM fluorescence; B3, PM fluorescence signal from cell in B1 after subtraction of ER fluorescence. (C) PM-integrated density for α4eGFP plus β2wt– and α4eGFP plus β4wt–transfected cells. Error bars show 99% confidence intervals. (D; 1 and 2) Plots showing number of pixels (y axis) versus fluorescence intensity (x axis) for either whole TIRF footprint (ER + PM) or the ER in α4eGFP plus β2wt– and α4eGFP plus β4wt–transfected cells. 3, the integrated density ratios of whole TIRF footprint to ER for α4eGFP plus β2wt and α4eGFP plus β4wt. Error bars are SEM. Number of imaged cells are indicated in parentheses. Bars, 5 µm.