Figure 3. P2X₇ R578Q is not expressed on the plasma membrane.

A, HEK293 stable cell lines were treated with the broad-spectrum protease proteinase K to detect the presence of the ∼30 kDa P2X₇ C-terminal tail that is liberated when the extracellular domain of P2X₇ is digested by protease when the receptor is localized at the plasma membrane. These data are representative of three experiments. B, HEK293 stable cell lines were treated with the cell-impermeable chemical cross-linker BS³ to detect P2X₇ localization at the cell surface. The P2X₇ monomers and putative dimers and trimers are indicated. The predicted molecular weight of human P2X₇ is ∼69 kDa and it contains several N-linked glycosylation sites that result in slower mobility in SDS-PAGE gels [15]. Thus, P2X₇ dimers are predicted to exhibit an apparent mass slightly greater than 140 kDa and P2X₇ trimers are predicted to exhibit an apparent mass slightly greater than 210 kDa. These data are representative of at least four experiments. C, HEK293 cells were stained with an anti-P2X₇ antibody or IgG isotype control, and the relative levels of surface-exposed P2X₇ WT and R578Q were determined by flow cytometry. The graph represents the average of three independent experiments. * p<0.006, ** p<0.007. gMFI = geometric mean fluorescence intensity.