Mouse Anti-HBEGF Recombinant Antibody (clone 2F7) (CAT#: HPAB-0119-YC)

Figure 1 Inhibition of ligand stimulation phosphorylation of EGFR in MDA-MB231 cells and HCT116 cells.

Cells were serum starved before addition of antibody and ligand. Cross-specific antibody (8d7 1f6 2f7, i.e. "2F7") was pre-incubated with recombinant HBEGF for 1 hour before addition to cells. Cells were stimulated for 20 mins before preparation of cell lysates. 30 μg of protein was loaded on SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was probed with anti pEGFR(Tyr1068)

Figure 2 Inhibition of ligand stimulation phosphorylation of EGFR in LoVo cells.

Cells were serum starved before addition of antibody and ligand. Cross-specific antibody (8d7 1f6 2f7, i.e. "2F7") was pre-incubated with recombinant HBEGF for 1 hour before addition to cells. Cells were stimulated for 20 mins before preparation of cell lysates. 30 μg of protein was loaded on SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was probed with anti pEGFR(Tyr 1068)

Figure 3 The results of a migration assay on MDA-231 breast carcinoma cell line assessed in the presence of 300 nM cross-specific monoclonal antibodies (8d7 1f6 2f7 i.e. “2F7” or 8d7 1c8 1e9 i.e. “1E9”) or isotype control.

The assay was left for 17 hrs before wound width was measured. The right hand panels showing representative images obtained in the absence of treatment and in the presence of 8d7 1f6 2b3 i.e. "2B3"

Figure 4 The results of an invasion assay of HUVEC cells through matrigel in the presence of 500 nM Bi-specific monoclonal antibody ((8d7 1f6 2f7 i.e. “2F7” or 8d7 1c8 1e9 i.e. “1E9” and negative control antibodies.

The cells were left for 24 hours before being stained by Hoescht and number of invaded cells per field of view counted.

Figure 5 Tube assay analysis of cross-specific antibody.

Huvec cells were seeded in the presence of cross-specific antibodies or control antibodies in wells coated with matrigel. Tube structure was analysed after 17 hrs. Images were taken and branching points around nodes counted. The lower panels show representative images with the upper panel illustrating in barchart format the number of branching points around nodes for untreated cells, cells treated with a control antibody and cells treated with the 8d7 1f6 2f7 antibodies.