Functional studies on LCLs. (A) Cytosolic ⁵⁵Fe incorporation in proband (P) and 2 controls (C1 and C2). Cells were incubated with 2 μM ⁵⁵FeAC and after 18 hours cpm determined in postmitochondrial fractions (PMFs). (B; top) Cytosolic aconitase activity in P and controls. (Bottom) Determination of IRP1 conformation by immunoblotting of native PAGE. Separated Fe/S- and apo-IRP1 proteins are indicated by arrows. β-actin (shown in panel D) was used to confirm equal protein content. (C; top) IRE-IRPs electro mobility shift assay (EMSA). β-ME (beta-mercaptoethanol) was used to evaluate the total binding activity. (Bottom) Western blotting after immunoprecipitation of LCL extracts using anti-IRP2. Equal immunoprecipitation was verified by revealing immunoglobulins (Ig's). (D; top) H ferritin determined by specific ELISA. (Bottom) Western blotting of TfR. Equal loading was verified by β-actin. The experiments were repeated 3 times. Representative pictures are shown. Error bars represent standard deviations. *P < .01.