Figure 2.
Expression of the NMD competent α1(S326fs328X) subunit minigene produced minimal mutant protein. A, Total lysates from HEK 293T cells cotransfected with β2 and γ2S subunit cDNAs and α1 subunit cDNAs (left) or minigenes (middle and right) with wild-type (wt) subunits (ratio of 1:1:1), mixed (mix) α1/α1(S326fs328X) or α1/α1(Y368X) subunits (0.5:0.5:1:1) and mutant (mut) α1(S326fs328X) or α1/α1(Y368X) subunits (1:1:1) with the Calcium phosphate precipitation method (Calcium). The total lysates were analyzed by immunoblot. Na+/K+ ATPase was used for a loading control. Wild-type α1 subunits migrated at ∼50 KD, α1(S326fs328X) subunits migrated at 37 KD, and α1(Y368X) subunits migrated at 43 KD. B, The relative amount of total wt, mix and mut α1 subunit protein from A was plotted. Mix 50 stands for the wild-type subunit level detected at 50 KDa in the mixed transfection condition, mix 37 or 43 stands for the mutant truncated subunits detected at 37 or 43 KDa in the mixed condition (**p < 0.01, ***p < 0.001 vs wild-type, ††p < 0.01 vs mix 50). C, HEK 293T cells, HeLa cells and rat cortical neurons were cotransfected with β2 and γ2S subunit cDNAs and wild-type α1, mixed α1/α1(S326fs328X) or mutant α1(S326fs328X) subunit minigenes (1:1:1) with Fugene transfection. In A and C, the green arrows indicate the mutant subunit. In C, U stands for untransfected. wt, mix, and mut are as described in A. D, Rat cortical neurons were transfected with β2 and γ2S subunit cDNAs and the wild-type α1 subunit minigene (left), the mutant α1 subunit minigene (S326fs328X) (middle) or the mutant α1(Y368X) subunit minigene (right) at a cDNA ratio of 1:1:1. After 6 d in culture, the neurons were permeabilized and stained with anti-monoclonal human α1 subunit antibody conjugated with fluorophore Alexa-647. In the insets, C refers to combined transmitted and fluorescent images, and F refers to fluorescent images. In C and D, neurons were transfected by nucleofection.