Figure 1 from Anti-HDGF Antibody Targets EGFR Tyrosine Kinase Inhibitor–Tolerant Cells in NSCLC Patient-Derived Xenografts

Characteristics of HDGF binding by humanized anti-HDGF antibody H3. A, Binding of native HDGF by anti-HDGF antibody H3. Recombinant H3 was immobilized on protein G agarose beads and used to capture HDGF in HEK293 lysate. Lane 1, H3 loaded-beads; lane 2, H3-beads plus HEK292 lysate; lane 3, captured protein probed with rabbit anti–HDGF antibody T221; lane 4, the same blot was striped and probed with goat anti–human IgG antibody. HDGF migrates at 37–42 kDa, larger than the calculated molecular weight of 26.7 kDa. B, Specificity of H3 recognition. Western blots of HEK293 parental (ctrl) or pooled HDGF knockout (T1, T2, and T3) cells probed with H3 or T221. The intensity of HDGF staining by H3 or T221 is reduced in the T1 and T2 knockout cells. C, Surface plasmon resonance sensorgram of the H3–HDGF interaction. The recombinant antibody was immobilized on the Biacore sensor chip, and GST-HDGF was used as the analyte. D, H3 epitope mapping. Bacterial expressing GST-HDGF fusion proteins were lysed, separated by SDS-PAGE, and probed with H3, followed by A2, an antibody that recognizes an epitope in HDGF PWWP domain. The full-length GST-HDGF fusion protein migrated with a molecular weight of 75 kDa (lane m), larger than the predicted size of 52 kDa. The GST-HDGF99 fusion protein (lane a) migrated at the predicted size of 37 kDa. The transition of H3 reactivity happens between constructs ending at residues 160 and 170, indicating that the H3 recognition epitope is in this region. E, H3 epitope and flanking sequence in mouse and human HDGF.