Figure 2.

(A) Determination of regions on FTL for interaction with preS and SCCA1. GST-tagged FTL deletion mutant proteins, each with deletion of one of the five α-helices (a to e), were allowed to bind to MBP-preS and His-SCCA1, and absorbed with Gutathione–Sepharose^TM beads. Proteins absorbed on Glutathione–Sepharose beads were subjected to Western blotting with anti-MBP antibody (detecting MBP-preS, upper), or with His-tag antisera (detecting His-SCCA1, down). (B) Verification of FTL-binding activity of N-terminal 1–11 amino acids of preS2. For the upper, pulldown assay was done by mixing GST-FTL with MBP-preS2 (lane 1) or MBP-preS2Δ (1–11) (deletion of N-terminal 1–11 amino acids of preS2, lane 2), and the proteins recovered by Glutathione–Sepharose beads were subjected to Western blot with anti-MBP antibody. For the down, pulldown assay was done by mixing GST-FTL with MBP (lane 1) or MBP-peptide (1–11) (MBP with the peptide of 11 amino acids from N-terminus of preS2, lane2), and the proteins recovered by Glutathione-Sepharose beads were subjected to Western blot with anti-MBP antibody.

Abbreviations: FTL, ferritin light chain; MBP, maltose binding protein; SCCA1, squamous cell carcinoma antigen 1; GST, glutathione-S-transferase.