Mutant FPNs do not coimmunoprecipitate with or affect the function of wtFPN. (A-B) 293T cells were doubly transfected with wtFPN-FLAG and either CD8, wtFPN–c-Myc, or mutant FPN–c-Myc as shown. Cells were lysed and immunoprecipitated (i.p.) with anti-CD8 (negative control), anti–c-Myc, anti-FLAG, or anti-FPNct. The precipitates were run on a gel, blotted onto membrane, and probed with anti–FLAG-HRP conjugate. Panel A shows cells transfected with CD8 and wtFPN-FLAG and cells transfected with wtFPN–c-Myc and wtFPN-FLAG. In both cases, precipitation with anti-FLAG results in a ferroportin band at around 70 kDa, revealed by probing the membrane with anti–FLAG-HRP. A much fainter band of the same size is precipitated by the anti-FPN polyclonal antisera. The same pattern of bands is seen when wtFPN-FLAG is coexpressed with c-Myc–tagged FPN mutants A77D, V162Δ, and C326Y (B). In each case no anti-FLAG reactive 70-kDa band is precipitated by anti–c-Myc, so that panel A indicates the wtFPN molecule does not form multimers, and panel B indicates that mutant FPNs also do not bind wtFPN. (C) 293T cells transfected with either CD8 alone, wtFPN-FLAG alone, or wtFPN-FLAG and mutant FPN–c-Myc together were lysed and tested for ferritin by ELISA as for Figure 1, except that this time no Tf was present in the media. Graph shows individual (•) and mean (horizontal bars) values for ferritin in ng/10⁷ cells. Error bars depict ± 95% CI. As before, cells expressing wtFPN have reduced ferritin compared with control transfectants expressing CD8. Cells expressing wtFPN along with FPN mutants A77D, V162Δ, and C326Y have a similar reduction in ferritin compared with wtFPN alone, indicating that the mutant FPNs do not interfere with the ability of wtFPN to make cells iron deficient.