FIGURE 3.

Effect of increasing mutant α1(AD) subunit cDNA on surface and total α1(AD) subunit protein expression. We transfected HEK293T cells with empty vector (negative control, shaded histograms) or 0.250 μg of β2 and γ2S subunits and 0.125 μg of α1^h subunit cDNA (hemizygous positive control, solid line histogram) or 0.250 μg of β2 and γ2S subunits, 0.125 μg of α1^r subunit, and increasing amounts of either α1(AD)^h or α1^h subunit cDNA. We measured the total (A and C) and surface (B and D) α1(AD)^h and α1^h subunit fluorescence by flow cytometry. A and B, dotted lines depict the fluorescence of cells transfected with 0.125 μg of α1(AD)^h subunits, and the dashed lines depict the fluorescence of cells transfected with 2 μg of α1(AD)^h subunits. C and D, we depicted the α1(AD)^h (●) and α1^h (○) fluorescence normalized to positive control for each mass of α1(AD)^h or α1^h subunit cDNA that we transfected. When measuring total α1(AD)^h and α1^h expression (C), increasing both α1(AD)^h and α1^h subunit cDNA caused linear increases in α1(AD)^h and α1^h subunit protein expression until α1^h subunit expression saturated when it was greater than 300% that of positive (pos) control. When measuring surface α1(AD)^h and α1^h expression (D), increasing α1^h subunit cDNA caused proportional increases in α1^h subunit expression, but increasing α1(AD)^h subunit cDNA resulted in very little change in surface α1(AD)^h subunit expression. E, we replotted the surface (surf) α1(AD)^h and α1^h subunit expression as a function of total expression. Both surface α1(AD)^h and α1^h subunit expression increased linearly with total expression (r² = 0.82 and 0.95, respectively, n ≥ 3). However, there was an 8-fold greater increased surface α1^h subunit expression than α1(AD)^h subunit expression for equal increases in total expression. To determine whether the presence of the wild type α1^r subunit facilitated the surface trafficking of α1(AD)^h subunits, we repeated experiments in the absence of the α1^r subunit and found no significant difference in the surface expression of the α1(AD)^h subunit in the absence of the α1^r subunit (n = 3, data not shown).