Cell-surface expression and intracellular localization of wtFPN and FPN mutants. (A) 293T cells were transfected with wtFPN–c-Myc and the mutant FPN–c-Myc variants indicated, and 2 days later stained for surface c-Myc tag and analyzed by flow cytometry for percentage of c-Myc–positive transfected cells and the MFI of the transfected cells (which is proportional to the amount of c-Myc expressed on the cell surface). The percentage of transfected cells did not vary significantly between wtFPN and mutant FPN constructs (70%-80% of cells were routinely c-Myc positive, not shown). However, the degree of fluorescence differed, with Y64N, C326Y, Q248H, N144D, and N144H all showing similar MFI comparable to wild type, while V162Δ, L170F, A77D, and G490D all express at levels less than 50% of wild type. (B) 293T cells were cotransfected with wtFPN-FLAG and either wtFPN–c-Myc or mutant FPN–c-Myc and stained with antibodies against the FLAG tag (red, i,iv,vii,viii) and the c-Myc tag (green, ii,v,viii,xi). The staining in i to iii shows that both FLAG-tagged and c-Myc–tagged wtFPN are expressed at the cell-surface membrane (arrowheads) as well as intracellularly. A77D FPN is located perinuclearly and not visible on the cell surface (arrowheads in v,vi) and does not colocalize with wtFPN, or alter the cell membrane staining of wtFPN (compare red with green in vi). Similarly, V162Δ locates predominantly intracellularly (arrowheads in viii,ix); however, x to xii show that C326Y FPN colocalizes with wtFPN at the cell membrane.