Reduced plasma membrane expression of Na/K pumps with FXYD1 and FXYD6. (A) Confocal image of representative oocytes following incubation in BODIPY FL-ouabain (1 µM). Note that preincubation of uninjected oocytes with unlabeled ouabain (UI+ouab) eliminates the signal observed in uninjected oocytes (UI). Fluorescence intensity was measured in two concentric circles (one including the plasma membrane signal) to subtract background fluorescence. The bar graph at the bottom summarizes results from two batches of oocytes (one batch for FXYD6) normalized to the fluorescence intensity measured in oocytes injected with α1β1 alone. (B) Representative Western blot of plasma membrane preparations from oocytes expressing α1β1 (lane 2), α1β1FXYD1 (lane 3), and FXYD1 alone (lane 4), 3 d after injection (14 oocytes in each condition). The top half was probed with the pan-α-antibody (a5, Developmental Studies Hybridoma Bank) and an anti-mouse secondary antibody (IRDye 800 CW goat anti-mouse; LI-COR). The bottom half was incubated with a polyclonal FXYD1 antibody (ab76597; Abcam) and an anti-rabbit secondary antibody (IRDye 680RD goat anti-rabbit; LI-COR). The bar graphs at the bottom summarize results from five membrane preparations from four oocyte batches, normalized to the intensity of α1β1 alone. Pump current activated by 4.5 mM K⁺, 145 mM Na⁺ in 28 α1β1 and 34 α1β1FXYD1 oocytes included in these membrane preparations were 219 ± 19 nA and 100 ± 10 nA (mean ± SEM), respectively.