Figure 1.

Exogenous Panx2 is localized intracellularly in stable expressing cell lines. (A) Schematic of the membrane topology of a Panx2 monomer. The Panx2 peptide sequence used to generate the carboxy terminal antibody used in this study is indicated in blue as well as an appended HA tag. (B) Western blot of a cell lysate from HeLa cells stably expressing Panx2-HA. Both the anti-Panx2 (polyclonal) and anti-HA (monoclonal) antibodies detect a ~80-kDa band corresponding to the molecular mass of Panx2-HA. (C–E) Images are single plane confocal micrographs. (C) In this HeLa cell line, immunofluorescence demonstrates almost complete overlap of anti-Panx2 (left image) and anti-HA antibodies (middle image). Anti-Panx2 is displayed in green, anti-HA in red and DAPI is blue in the merged images for (C) (right column). (D) MDCK cells stably expressing Panx2-4Cys stained with FlAsH (green) and the anti-Panx2 antibody (red) also show almost complete overlap of the two fluorescent signals. (E) In this panel, no primary antibody was used in the initial incubations (control, left image), while ReAsH staining (red) confirmed the presence of Panx2-4Cys (middle image).