Interaction studies of panx1a and the Y205A mutant using FRET and pull-down assay. (A) Fluorescent image showing a Neuro 2a cell transfected with panx1a-DsRed and Y205A-EYFP. Scale bar: 10µm. (B) FRET efficiencies. WT–WT (membrane) (n = 103) indicates panx1a-DsRed interaction with panx1a-EYFP using selected cell membrane regions as a positive control. WT–WT (intracellular) (n = 100) indicates panx1a-DsRed interaction with panx1a-EYFP using selected regions inside the cell. WT-Y205A (n = 63) indicates panx1a-DsRed interaction with Y205A-EYFP inside the cell. Y205A-Y205A (n = 50) indicates Y205A-DsRed interaction with Y205A-EYFP inside the cell. DsRed-EYFP indicates DsRed interaction with EYFP inside the cell as the negative control. The dotted line represents the FRET efficiency threshold of 1.4%. (C) His60 Ni pull-down using cells double-transfected with panx1a-dTomato-His and panx1a-EYFP, Y205A-dTomato-His and panx1a-EYFP, or dTomato-His with panx1a-EYFP. Cell lysates on the left show expression levels before pull down. Elutions on the right show proteins recovered by pull down. Anti-GFP and anti-His antibodies detected eluted proteins. An anti-ß-actin antibody served as a loading control. ****p < 0.0001, N.S., not significant.
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