Fig. 10. Ouabain inhibits proximity events between the α1-subunit of the Na,K-ATPase and FGF2 in a cellular context.

DuoLink assays (PLA®; Sigma-Aldrich) were conducted to quantify proximity of the α1 chain with FGF2 in a cellular context as described previously¹⁵. Cells were incubated with ouabain for 2 h at the concentrations indicated. Cells were fixed with acetone. Nuclei were labeled with Sytox green (Life Technologies) and cells were imaged by confocal microscopy. For further details, see Methods. a Representative example of mock-treated HeLa cells. Scale bar = 10 µm. b Selected area of panel a at a higher magnification. DuoLink proximity events (red dots) in the vicinity of the plasma membrane are labeled with white arrowheads. c Representative example for cells treated with 50 µM ouabain. d Quantification, normalization, and statistical analysis of α1/FGF2 proximity events in the absence and presence of ouabain at the concentrations indicated. Images were analyzed with the DuoLink Image Tool Software (Olink Bioscience). Background signals were subtracted and data were normalized relative to the mock control. Data are shown as mean ± SD (n = 5). P value for (a) was 0.1124; p value for (b) was 0.0381. ***p ≤ 0.001.