FIGURE 4.

Protein expression of mutant EDA1 in transfected cell. (a) HEK293T cells were transfected with vectors encoding mutant or wild‐type soluble Myc‐tagged EDA1 protein, supernatants, and cell lysates were separately analyzed by western blotting, β‐tubulin was used as a loading control. The bands showed that wild‐type EDA1 can produce intracellular and extracellular proteins, the weaker bands of p.F379S and p.[R289P;S290C] mutations in the cell lysates showed decreased intracellular protein expression, and the lack of band of p.F379S and p.[R289P;S290C] mutations in the supernatant revealed these mutations fully impaired expression of soluble EDA1. (b) HEK293T cells were transfected with WT or mutant EDA1 protein vector contained an N‐terminal Myc tag. Nucleus was labeled with DAPI (blue), cells were stained with anti‐Myc antibody, followed by Alexa Fluor 488‐conjugated secondary antibody. The decreased fluorescence intensity of p.F379S and p.[R289P;S290C] mutations showed decreased intracellular EDA1 protein expression. Scale bar: 5 μm. WT, wild type. Data shown are representative of three experiments