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Fig. 10. Panx1 and Panx3 assemble into functional single-membrane channels in 293T cells but do not form functional intercellular channels in N2A cells. (A) Fluorescent micrographs of wild-type (WT) and pannexin-expressing 293T cells after sulforhodamine B dye uptake (left column: 20x objective). The middle column denotes the corresponding phase-contrast images, which includes the background uptake of the large control dye, dextran-rhodamine (inserts). (Right column) In parallel experiments, immunolabeling for pannexins (red) and counterstaining with Hoechst dye (blue) revealed the high pannexin transfection efficiency (63x oil objective). (B) GJIC-deficient N2A cells were engineered to transiently express Cx43-GFP, Cx26-GFP, Panx1-GFP or Panx3-GFP, or to co-express Panx1 or Panx3 and DsRed. Isolated N2A cell pairs with clear green or red fluorescence were chosen for dual whole-cell patch-clamp recordings 48 hours later to measure intercellular junctional conductance. The expression of Cx43-GFP and Cx26-GFP resulted in robust electrical coupling conductance between N2A cells, which was significantly higher than the coupling observed in paired control wild-type N2A (wt N2A) cells. However, both Panx1- and Panx3-expressing N2A cells exhibited no significant increase in electrical coupling above what was observed in wild-type N2A cells. Data were presented as mean±s.e.m.; **P<0.01. The number presented over each column represents the n value for each experimental condition.
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