Figure 5. Inhibition of 14-3-3/ligand interaction does not affect oligomerization between α-subunits of Nav1.5.

Figure 5. Inhibition of 14-3-3/ligand interaction does not affect oligomerization between α-subunits of Nav1.5. Difopein and its mutant did not affect co-immunoprecipitation between α subunits of Nav1.5. (a) Representative immunoblot of the total lysate ("INPUT") and FLAG-specific immunoprecipitated fraction ("IP: FLAG") of tsA201 cells transiently expressing Nav1.5 with empty vector, difopein, or mutant of difopein. Overexpressed YFP-tagged difopein and its mutant were revealed with an anti-GFP antibody. Endogenous α-actin was used as a negative control for co-immunoprecipitation with Nav1.5. (b) Intensity of co-immunoprecipitated Nav1.5-HA was normalized to the intensity of the total Nav1.5-HA divided by the intensity of α-actin. Data are presented as mean ± SEM from three biological replicates and are normalized to the control condition ("+ empty vector"). Individual p-values, calculated with one-sample two-tailed t-test with hypothetical mean value=1, are indicated in the panel. (c) Schematic illustration of NanoBiT assay. Background signal was determined by co-expression of N- or C-termini LgBiT-Nav1.5 with non-interacting control, SmBiT-HaloTag. (d, e, f, and g) In living cells difopein did not modify the level of Nav1.5 dimers. Results of the NanoBiT assay are presented as relative intensity of luminescence (indicating the level of protein-protein interactions) normalized to fluorescence (indicating the number of cells) in tsA201 cells transiently transfected with SCN5A constructs. Each dataset was normalized to its relevant background. Different variations between Nav1.5 N- and C-termini interactions were tested. Since difopein and its mutant were tagged with YFP, we also included control by co-transfection with GFP. NT, non-transfected. Data are presented as mean ± SEM from three biological replicates. The multiplicity adjusted p-values, calculated with one-way ANOVA and post hoc Tukey's multiple comparisons, are indicated in each panel.