Afuco™ Anti-Human ENG ADCC Recombinant Antibody (TRC105), ADCC Enhanced (CAT#: AFC-220CL)

Figure 1 TRC105 promotes steady-state and ligand-induced Smad2/3 activation while inhibiting Smad1/5/8 signaling.

Western blot analyses show endogenous expression of phosphorylated (p-) Smad2/3 (activated), total Smad2/3, p-Smad1/5/8 and total Smad1 in cell lysate following a 24 h treatment with TRC105 (0–2 μg/ml) in HMEC-1 (a) and HUVEC (b). β-actin levels indicate equal loading of lysates in both cell types. (c) Western blot shows Smad1/5/8 and Smad2/3 activation in HMEC-1 under no treatment, TGF-β1 (100 pM) and TRC105 (0.2 μg/ml) plus TGF-β (100 pM) for 30 min. Lower panels show total Smad1 and Smad2/3 from the same cell lysate. (d) Western blot shows Smad1/5/8 activation in response to BMP-9 treatment (30 min) in HUVEC in the presence or absence of TRC105 (0.2 μg/ml) for 30 min. Data are representative of at least three independent experiments.

Kumar,S. Pan,C.C. Bloodworth, J.C.,Nixon, A.B.,Theuer, C.,Hoyt,D.G., & Lee, N. Y. (2014). Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene, 33(30), 3970.

Figure 2 TRC105 does not induce endothelial growth arrest or apoptosis.

(a) MTT assay showing the HUVEC growth pattern following treatment with either control IgG or TRC105 (2 μg/ml) for 12, 24, 48 h (left graph). A parallel MTT assay showing the effects of control and stable endoglin depletion through shRNA (shEng) in HMEC-1 (right graph). *P=0.001 comparing control versus shEng at 24 h. (b) HMEC-1 cells were treated with different concentrations of TRC105 (0–2 μg/ml) and TGF-β1 (400 pM) for 24 h, and assessed for cytochrome c release via immunofluorescence. Shown are representative images of cells treated with TRC105 and TGF-β1. Arrow identifies a cell from which cytochrome c was released. Data are mean±s.d. of at least 30 cells counted for each condition (**P<0.01 compared with control 0 μg/ml). (c) The western blot shows caspase-9 and -3-cleaved products upon treatment of HMEC with either IgG control or TRC105 (2 μg/ml) for 16 h.

Kumar,S. Pan,C.C. Bloodworth, J.C.,Nixon, A.B.,Theuer, C.,Hoyt,D.G., & Lee, N. Y. (2014). Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene, 33(30), 3970.

Figure 3 Endoglin-targeting Abs enhance sEng production.

(a, b) Western blot of sEng immunoprecipitated from conditioned media (Abs P3D1 or H-300) after 24 h treatment with TRC105 (0–2 μg/ml) in HMEC-1 (a) and HUVEC (b) (top panels). Middle and lower panels show endogenous expression of membrane-anchored full-length endoglin, MMP-14 and β-actin in cell lysates. Graphs represent the density ratio of sEng to β-actin. (c) Endogenous sEng immunoprecipitated from the conditioned media upon 24 h treatment with indicated Abs directed against different epitopes on endoglin extracellular domain (200 ng/ml). Lower panels show expression of full-length endoglin, MMP-14 and β-actin in cell lysates. All of the western blots are representative of at least four independent experiments.

Kumar,S. Pan,C.C. Bloodworth, J.C.,Nixon, A.B.,Theuer, C.,Hoyt,D.G., & Lee, N. Y. (2014). Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene, 33(30), 3970.

Figure 4 TRC105 enhances endoglin/MMP-14 association at the cell surface.

(a) Western blot analysis shows co-immunoprecipitation of endoglin and MMP-14. COS-7 cells expressing HA-tagged MMP-14 (MMP), endoglin (Eng), MMP and Eng in the presence or absence of TRC105 (200 ng/ml) pretreatment for 10 min. Lysates were immunoprecipitated with MMP-14-specific Ab and immunoblotted for co-immunoprecipitated endoglin, MMP-14, total endoglin, and β-actin. (b) Western blot analysis shows co-immunoprecipitation of Eng and MMP-14 following immunoprecipitation with HA-Ab (MMP-14). (c) Cell surface biotinylation assay was performed in COS-7s expressing HA-tagged MMP-14 (MMP), Eng, MMP and Eng in the presence or absence of TRC105 pretreatment (200 ng/ml) at 4 °C. Cell lysates were prepared following biotinylation, and MMP-14 was immunoprecipitated with HA-Ab. The western blot shows biotinylated endoglin and MMP-14 detected with streptavidin-horseradish peroxidase (HRP). Data are representative of at least three independent experiments. (d) HUVECs were treated with TRC105 alone (200 ng/ml), MMP-14 blocking Ab alone (2 μg/ml), or co-treated for 24 h. Graph represents a densitometry analysis of sEng present in each condition as a ratio compared with no treatment. Each error bar represents the s.d. derived from three independent experiments normalized to no treatment. *P<0.001, **P=0.0014, ***P<0.001.

Kumar,S. Pan,C.C. Bloodworth, J.C.,Nixon, A.B.,Theuer, C.,Hoyt,D.G., & Lee, N. Y. (2014). Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene, 33(30), 3970.

Figure 5 TRC105 promotes MMP-14 gene expression in HUVEC.

(a) Cells treated with TRC105 (200 ng/ml) for 24 h were quantified by SYBR green based quantitative PCR and analyzed by delta-delta-CT (ddCT) methods using18S rRNA as internal control. Fold changes were calculated by setting the mean fractions of untreated cells as one. Bars indicate mean±s.d. in cells from TRC105-treated and -untreated cells. (b) Graph shows the effect of the ALK5 inhibitor (SB431542, 5 μM) and/or TRC105 (200 ng/ml) for 24 h on MMP-14 gene expression in HUVEC. Inset of western blot shows endogenous expression of sEng immunoprecipitated from the conditioned media of the same cells that were used to isolate RNA for MMP-14 gene expression study. (c) Graph shows MMP-14 gene expression upon treatment for 24 h with TRC105, JNK inhibitor (SP600125, 5 μM) and TRC105 with JNK inhibitor. (d) Western blot analysis shows TRC105 concentration-dependent phosphorylation (activation) of JNK (upper panel) with t (lower panel) as loading control. *P<0.05, **P<0.001.

Kumar,S. Pan,C.C. Bloodworth, J.C.,Nixon, A.B.,Theuer, C.,Hoyt,D.G., & Lee, N. Y. (2014). Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene, 33(30), 3970.

Figure 6 TRC105 and sEng induce cell migration.

(a) HUVEC and (b) HMEC-1 were plated on transwells coated with 0.02% gelatin and treated with TRC105 (0 to 2 μg/ml) in growth media for 16 h. Cells that migrated to the bottom side of the membrane were fixed, stained for their nuclei and imaged for counting by using ImageJ software. (c) The graph represents the effect of TRC105 treatment (200 ng/ml with or without MMP-14-neutralizing Ab (1:500 v/v)) treatment on migration of HMEC-1. (d) The graph shows migration of HMEC-1 expressing the secreted endoglin extracellular domain (Eng-ECD), treated with or without TRC105 or MMP-14 Ab. *P<0.05, **P<0.001, ***P<0.08. Relative cell migration is represented as a percentage compared with untreated cells, from triplicates for each of the three independent experiments. The error bars indicate the s.e.m. of the percentage of the cells that migrated.

Kumar,S. Pan,C.C. Bloodworth, J.C.,Nixon, A.B.,Theuer, C.,Hoyt,D.G., & Lee, N. Y. (2014). Antibody-directed coupling of endoglin and MMP-14 is a key mechanism for endoglin shedding and deregulation of TGF-β signaling. Oncogene, 33(30), 3970.