Fig. 3

Our newly developed anti-HER3 mAb 4A7 recognizes cell membrane HER3, and exhibits profound activity to block HRGβ1-, but not SDF-1-induced TNBC cell migration. A, Our anti-HER3 mAb 4A7 was produced and purified as described in the materials and methods. 4A7 binding to membrane HER3 on SKBR3.B3.1 cells was examined by flow cytometry analysis. Staining with 2nd Ab only was used as a negative control. 10,201-MM01, a commercially available anti-HER3 mAb was used a positive control. Quantification of the positive staining with either 10,201-MM01 or 4A7 from three independent experiments was shown in the bar graphs. B, Equal amount of total protein extracts of HCC1806 cells were either examined by western blot analyses of HER3 and IGF-1R (Input) or subjected to IP (IP: Ab) using a control IgG or 4A7 and followed by western blot analyses of HER3 and IGF-1R. C-D, The specificity of 4A7 against HER3 was confirmed by its capability to block HRGβ1-induced promotion of TNBC cell migration. HCC1806 cells seeded in 96-well plates were pre-incubated with mitomycin C (2 µg/ml) prior to the scratch assays. The cells were then untreated or treated with HRGβ1 (50ng/ml) or HRGβ1 plus 4A7 (20ug/ml) and monitored by an IncuCyte system for cell migration (C). The same migration assays were performed with MDA-MB-231 cells in the presence of HRGβ1 (50ng/ml) or SDF-1 (200ng/ml) or their combinations with 4A7 (D)