Analysis of the specificity of the selected mAbs and their molecular targets.

(A) IIF analysis performed on L-CD71⁺ fibroblasts reveals that the mAbs do not cross-react with TFR-1. Selected mAbs are representative for groups A (G/14E8) and B (G/1C11). The polyclonal antiserum features the same reactivity pattern. The same cells are clearly stained by the anti–TFR-1 mAb CB26. Gray profiles show the staining obtained by using an irrelevant antibody. X-axis, fluorescence intensity/cells; y-axis, number of cells registered/channel. (B) Transfectants and cell lines were lysed, run in 6% SDS-PAGE under reducing conditions, and transferred to a nitrocellulose membrane. G/14E8 mAb reacted with a dominant band of approximately 90 kDa in HepG2, in NIH-3T3/TFR-2/4B1, and in K562. A minor band of −105 kDa was observed in K562 cells (arrow). Lysates from NIH-3T3 mock-transfected cells and from HL-60 and L-CD71⁺ fibroblasts did not show any detectable bands. The reaction was revealed using a CDP-STAR chemiluminescence reagent, and the membranes were subjected to autoradiography. (C) RT-PCR analysis of a TFR-2 fragment confirmed the presence of the relevant message in HepG2, in NIH-3T3/TFR-2/4B1, and in K562 cells. Mock-transfected NIH-3T3 cells, HL-60 cells, and L-CD71⁺ fibroblasts did not show any message for TFR-2. GAPDH is shown as a control of RNA quality.