Figure 4.

Ligation of the Eph ECD α-thioester with synthetic peptides. (A) The reactive α-thioester generated at the C terminus of the EphB4/A3 ECD was ligated to a peptide with FLAG-tag sequence and an N-terminal cysteine residue (CGGGDYKDDDDK). The ligation reaction was resolved on SDS-PAGE and visualized using anti-Flag antibody on a Western blot as shown. (Mw) Molecular weight marker (in kilodaltons). The blot shows a signal for the ligated products but not for the controls, i.e., receptor thioesters before the ligation, and the small peptide by itself. (B) Modification of Eph ECD with Histidine-tag. EphA3 ECD α-thioester was ligated with a FLAG/His-tagged peptide (CGGGDYKDDDDKHHHHHH, MW = 2.1 kDa). The ligated product binds to the Ni²⁺ chelating beads (B) and was resolved on an SDS-PAGE. The results were visualized with an anti-FLAG antibody as shown on the Western blot. (Mw) Molecular weight marker (in kilodaltons). (Lane 1) EphA3 ECD α-thioester mixed with peptide. The ligated product is confirmed by signal on the blot. Lane 2 (peptide by itself) and lane 3 (EphA3 ECD α-thioester by itself) show no signal on blot. (Lanes 4–11) Controls (lacking different ligation components) showing supernatant (S) and beads (B) after binding to the Ni²⁺ chelating beads. Only EphA3 ECD α-thioester mixed with the peptide (lanes 12,13) gives a ligated product that binds to the beads and shows a signal on the blot. (C) Biotinylation of Eph ECD with hydrophilic peptide. EphA3 ECD α-thioester was ligated with FLAG-tag peptide that is biotinylated at its C terminus (CGGGDYKDDDD-K-Biotin). The ligated product was resolved on SDS-PAGE gel and analyzed with AP-conjugated Streptavidin (I) or anti-FLAG antibody (II) in the Western blots as shown. (Mw) Molecular weight marker (in kilodaltons). The ligated product is confirmed by signal on the blot with both methods of staining while controls give no signal. (D) Biotinylation of Eph ECD with hydrophobic peptide. EphA3 ECD α-thioester was ligated with a hydrophobic peptide, derived from the TM Eph region, that is biotinylated at its C terminus (CLALIAGTAVK-Biotin). The ligated product was resolved on SDS-PAGE and analyzed with AP-conjugated Streptavidin on Western blot. (Mw) Molecular weight marker (in kilodaltons). (Lane 2) The ligated product is confirmed by signal on the blot; (lanes 1,3) controls lacking either the peptide or the Eph thioester.