Fig. 1.

Unx1 is a membrane protein with a conserved topology among members of the large-pore channel family. (A) Superresolution analysis of Unx1-EGFP localization in HeLa transfectants. Anti-α1 Na⁺/K⁺ATPase antibody was used as membrane marker, whose immunofluorescence was colocalized with that of EGFP fused to Unx1. The yellow box in the merge (bottom 6.5× amplification of rectangle denoted in the above panel) corresponds to the area of evaluation of the Pearson correlation index. (B and C) HEK293T cells were cotransfected with EGFP-Unx1 and pmRFP fused to ß-1-4 galactosyltransferase (Golgi) (B) or to CgA (C) to assess whether the Unx protein traffics to the plasma membrane. (D) Confocal immunofluorescence of HeLa cells transfected with the pcDNA3.1-Unx1-EGFP vector using rabbit anti-Unx1 antibodies (Abs#1 or #2) or anti-EGFP. Cy3 anti-rabbit signals were only detected in permeabilized cells treated with 0.01% Triton X-100, confirming the presence of intracellular epitopes. (Scale bar, 20 µm.) (E) Western blot analysis of total proteins evaluated with anti-EGFP antibody (1:1,000) from Cx45^−/− HeLa cells 24 h after transfection with different Unx1 constructs, as shown in the figure. Anti- a-tubulin was used as the loading control. (F) Schematic representation of the proposed topology for Unx1 in the cell membrane, highlighting the N and C termini in intracellular space.