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. 2014 Jul;144(1):81–104. doi: 10.1085/jgp.201411169

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© 2014 Xu et al.

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Figure 8. — Evaluation of total, surface, and intracellular fractions of P2X4-pHluorin123 receptors in HEK-293 cells. (A) The schematic shows P2X4-pHluorin123 receptors expressed on the PM and within vesicles. The P2X4-pHluorin123 receptors were fluorescent in neutral environments, quenched in the acidic compartments of intracellular vesicles, and unquenched in all compartments when the cells were bathed in NH₄Cl. (B–D) Representative images of HEK-293 cells shown under the indicated conditions. Note, as shown in the cartoon in A, that the fluorescence intensity was quenched in acidic buffers and increased in NH₄Cl. The inset shows a zoomed-in version of the cell in NH₄Cl. Under this condition, numerous vesicles became visible. (E) Average graph showing how the fluorescence of HEK-293 cells expressing P2X4-pHluorin123 changes under the indicated conditions. (F) The top two panels show the FWHM of vesicles that became visible in the presence of NH₄Cl. The graphs reveal a range of vesicle sizes (FWHM) and intensities, perhaps representing different loads of P2X4-pHluorin123. The bottom bar graph shows the proportion of P2X4-pHluorin123 receptors on the cell surface, within intracellular compartments, and within other compartments of HEK-293 cells. To generate this graph, the data were analyzed as described in Materials and methods using data from experiments such as those shown in E. Error bars represent mean ± SEM.