recombinant human hbegf  (R&D Systems)

Journal: Science signaling

Article Title: A ligand-receptor interactome platform for discovery of pain mechanisms and therapeutic targets

doi: 10.1126/scisignal.abe1648

Figure Lengend Snippet: (A to D) Intracellular Ca2+ concentration in mouse male and female DRG neurons in response to HBEGF (A, analyzed in B) or HBEGF with the EGFR antagonist lapatinib (C, analyzed in D). N = 306 and 595 neurons, respectively. (E to J) Mechanical sensitivity to von Frey filaments, as inferred from paw withdrawal thresholds and grimacing, in male ICR mice (E and F) and female C57BL/6 mice (G and H) one hour after hindpaw injection with saline or 50 ng HBEGF. Pooled data for all male and female are displayed in (I and J). N = 5 to 11 mice each, as noted in the figure. * p <0.05, ** p<0.01, *** p<0.001 by two-way ANOVA with Bonferroni post test, or t-test for effect size. Sample sizes are shown in the figure. Horizontal bars or boxes represent the mean, and error bars represent SEM.

Article Snippet: Experimental Reagents Recombinant human HBEGF (259-HE) was purchased from R&D Systems and reconstituted to 50 μg/ml in 0.1% BSA.

Techniques: Concentration Assay, Injection, Saline

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: Affymetrix probe list

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF is decreased in patients with NB and correlates with survival. A) HBEGF expression in the microarray meta-dataset in benign neuroblastic tumors (ganglioneuroma/ganglioneuroblastoma) or NB tumors. ****P < 0.0001 (Mann-Whitney U test). B) Immunofluorescence in neuroblastoma specimens for HBEGF (green). DAPI nuclear stain in blue. Original magnification, ×40. Scale bar, 50 μM. C) HBEGF expression in patients with NB by stage. P < 0.0001, Kruskal-Wallis test. **P < 0.01, ***P < 0.001, ****P < 0.0001 Mann-Whitney U test for intergroup comparisons. D) Event-free survival (EFS) in patients with NB with low (bottom 50%; red) and high (top 50%; blue) HBEGF expression in the GSE49710 dataset. E) HBEGF expression in the GSE49710 dataset in nonamplified (NA) or MYCN-amplified (Amp) NB tumors. ****P < 0.0001 (Mann-Whitney U test). Box plots are presented as median (horizontal bars) and interquartile range (boxes).

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Microarray, MANN-WHITNEY, Immunofluorescence, Staining, Amplification

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF promotes neuroblast differentiation in NB cells. A) Western blot for differentiation markers after 72 h of HBEGF treatment in BE2 and SK-N-AS. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. B) Quantification of NF160 densitometry normalized to β-actin from 3 independent Western blots (5Y, BE2) or 8 independent Western blots (SK-N-AS) after 72-h treatment with 1 ng/ml HBEGF and presented as means ± sem. P < 0.001 (1-way ANOVA). *P < 0.05, **P < 0.01, ****P < 0.0001, 1-sample Student’s t test. C) Representative phase-contrast images of BE2 cells after 72 h of treatment with HBEGF. Arrows identify long neurites. Original magnification, ×10. Scale bar, 100 μM. D) Quantification of neurite length using NeuronJ after 72 h of treatment with HBEGF from 3 independent experiments. P < 0.01 (1-way ANOVA). *P < 0.05, 1-sample Student’s t test. E) Western blot for differentiation markers after 72 h of HBEGF (0.5 or 1 ng/ml), FGF2 (1 or 10 ng/ml), or ATRA (1 or 10 μM). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. F) Western blot for differentiation markers after 72 h HBEGF (0, 0.25, 0.5, 0.75, 1, 2 ng/ml) and a neutralizing HBEGF antibody (nAb; 0.0075, 0.015, 0.03, 0.05, 0.1, or 0.5 μg/ml). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. G) Western blot for β3-tubulin and HBEGF in SHEP stably expressing an NTC shRNA or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for β3-tubulin normalized to β-actin is shown as the percentage of control. H) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right).

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Microarray

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: Schwannian stroma-derived HBEGF promotes neuroblast differentiation. A) Immunofluorescence in NB specimens using HBEGF (green) and S100 schwannian stroma (red) antibodies. DAPI nuclear stain in blue. Original magnification, ×40. Scale bar, 50 μM. B) Western blot for differentiation markers in 5Y after 72 h of coculture or treatment with conditioned medium from SHEP stably expressing an NTC shRNA construct or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for NF160 normalized to β-actin is shown as the percentage of control.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Immunofluorescence, Staining, Western Blot, Stable Transfection, Expressing, shRNA, Construct, Control

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HSPGs and EGFR interact to promote HBEGF-mediated neuroblast differentiation. A) Western blots for differentiation markers after 72-h treatment with 10 ng/ml soluble (s)TβRIII or sCD44 or 100 ng/ml sGPC1, sGPC3, or sSDC3 in the absence or presence of 0.5 ng/ml HBEGF in 5Y cells. B) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, a dosecourse of HBEGF in SK-N-AS after 96-h TβRIII knockdown. C) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, 0.5 μg/ml heparin, ODSH, 2-O desulfated heparin (2DES), 6-O desulfated heparin (6DES), or N desulfated heparin (NDES) in the absence or presence of 0.5 ng/ml HBEGF in SK-N-AS. D) Densitometry for NF160 normalized to β-actin is shown as the percentage of control, EGFR inhibitors for 72 h followed by 48 h of 1 ng/ml HBEGF treatment. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. E) In situ proximity ligation assay in SK-N-AS after 5 min of treatment with 1 ng/ml HBEGF or 1 ng/ml EGF. Original magnification, ×40. Scale bars, 50 μM. Normalized TβRIII/EGFR complexes per cell (75–100 cells/condition) from 6 independent experiments. P < 0.001 (1-way ANOVA). *P < 0.05, ****P < 0.0001, 1-sample Student’s t test. F) Analysis of event-free survival in the GSE49710 dataset stratified by the top and bottom 50% for HBEGF, then TGFBR3, then EGFR expression (left) and analysis of event-free survival stratified by the top and bottom 12.5% for HBEGF, then TGFBR3, then EGFR expression (middle) compared with stratification by MYCN amplification status (right). NA, nonamplified.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Knockdown, In Situ, Proximity Ligation Assay, Expressing, Amplification

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF induces neuroblast differentiation via ERK and STAT3 signaling and up-regulation of ID1. A) Western blot in SK-N-AS for phosphorylated and total STAT3 or ERK1/2 after 72 h treatment with 1 ng/ml HBEGF. Densitometry for phosphorylated STAT3 or phosphorylated ERK1/2 normalized to β-actin is shown as the percentage of control. B) Western blot for ID1 in BE2 and SK-N-AS after 72 h treatment with a dosecourse of HBEGF. Densitometry for ID1 normalized to β-actin is shown as the percentage of control. C) Western blot for ID1 in SHEP stably expressing an NTC shRNA or shRNA to HBEGF (shHBEGF #1, #2). Densitometry for ID1 normalized to β-actin is shown as the percentage of control. D) Western blot in SK-N-AS after 96 h ID1 knockdown and 72 h HBEGF treatment. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. E) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right). F) Western blot for differentiation markers and ID1 after 24 h cotreatment with 1 ng/ml HBEGF and the indicated doses of U0126 or CI-1040. Densitometry for NF160 normalized to β-actin is shown as the percentage of control. G) Western blot for differentiation markers after 72 h expression of an empty-vector control (EV) or a dominant negative STAT3 (DN STAT3) construct and 48 h treatment with 1 ng/ml HBEGF or 48 h treatment with ruxolitinib and 24 h treatment with 1 ng/ml HBEGF. Densitometry for NF160 normalized to β-actin is shown as the percentage of control.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Microarray, Plasmid Preparation, Dominant Negative Mutation, Construct

Journal: The FASEB Journal

Article Title: Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

doi: 10.1096/fj.201600828R

Figure Lengend Snippet: HBEGF suppresses neuroblast proliferation. A) Proliferation index from 3 (5Y) or 4 (SK-N-AS, BE2) replicates (means ± sem) of thymidine incorporation after HBEGF treatment for 24 h (SK-N-AS), 48 h (5Y), or 72 h (BE2), normalized to untreated control. P < 0.0001 (1-way ANOVA). **P < 0.01, ***P < 0.001 (1-sample Student’s t test). B) Western blot for p21 after 72 h of HBEGF treatment in BE2 and SK-N-AS. Densitometry for p21 normalized to β-actin is shown as the percentage of control. C) Linear regression analyses using the microarray meta-dataset (left) or the GSE49710 dataset (right). D) SK-N-AS NTC and SK-N-AS shHBEGF#1 subcutaneous xenograft. Tumors were measured at d 19 using calipers, and this measurement was used to calculate the fold change in tumor growth after 21, 24, 26, and 31 d. **P < 0.01, Kruskal-Wallis to compare curves.

Article Snippet: Recombinant soluble human HBEGF (259-HE), type III TGF-β receptor (TβRIII) (242-R3), glypican (GPC)1 (4519-GP), GPC3 (2119-GP), syndecan (SDC)3 (3539-SD), and CD44 (3660-CD) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Control, Western Blot, Microarray

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Upregulation of MMP2 at low O2, and its effects on HBEGF and cell survival. (a) Expression arrays for MMP-related proteins incubated with extracts of HTR-8/SVneo cells cultured at 20% (left) or 2% (right) O2. Arrows indicate elevated MMP2 at 2% compared with 20% O2. The key below indicates the location of duplicate antibody probes for each protein according to the coordinates shown, including both positive (Pos) and Negative (Neg) controls. (b) Western blots of molecular weight standards (Ladder), recombinant proMMP2 or proMMP9 zymogens (Control), and extracts of TB cells cultured at 20% or 2% O2, as indicated. The upper blot was labeled with anti-MMP2, the middle blot was labeled with anti-MMP9, and lower blot was labeled with anti-β-actin. (c) MMP2 quantified by ELISA in TB cells cultured 0–4 h at 2% O2. (d) HBEGF quantified by ELISA in TB cells treated during culture for 4 h with the indicated MMP inhibitor or vehicle, and concentration of O2. (e) Apoptosis was quantified in TB cells cultured as in D using the TUNEL assay. *P<0.05, compared with the control (0 h, vehicle/20% O2), n=3

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Expressing, Incubation, Cell Culture, Western Blot, Molecular Weight, Recombinant, Control, Labeling, Enzyme-linked Immunosorbent Assay, Concentration Assay, TUNEL Assay

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Expression of MMP2 and HBEGF at 2% O2 in HTR-8/SVneo cells. MMP2 (a) and HBEGF (b) mRNA were measured by qPCR after culture for indicated time at 2% O2. HBEGF protein measured by ELISA in extracts of TB cells cultured at the indicated concentrations of α-amanitin and O2 (c) and in cellular extracts of TB cells cultured for 4 h at either 20% or 2% O2 in the presence of 10 nM recombinant MMP2 (rMMP2) and α-amanitin, as indicated (d). *P<0.05, compared with no treatment/vehicle/20% O2, n=3

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Expression of HBEGF in presence of CoCl2 in HTR-8/SVneo cells. (a) Western blots of HIF1A (~ 93 kDa), HIF2A (~96 kDa) and β-actin (~45 kDa) in lysates of TB cells treated at 20% O2 or 2% O2 for 4 h, or at 20% O2 in the presence of 250 μM CoCl2 for 0.5–2 h, as indicated in the key below gel images. HIF1A (b) and HIF2A (c) proteins measured by ELISA in extracts of TB cells treated at 20% O2 or 2% O2 for 4 h, or at 20% O2 in the presence of 250 μM CoCl2 for 0.5–2 h. (d) HBEGF protein measured by ELISA in extracts of TB cells cultured at the indicated concentrations of α-amanitin and CoCl2 at 20% O2. *P<0.05 compared with no treatment control, n=3

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Effect of HIF1A and HIF2A knockdown on regulation of HBEGF by O2. HIF1A (a) and HIF2A (b) protein measured by ELISA in extracts of HTR-8/SVneo cells cultured for 48 h at 20% O2, with or without 50 pM of either scrambled siRNA or siRNA for HIF1A and HIF2A. Afterwards, culture was continued for 4 h at 20% O2, or at 2% O2 with vehicle, scrambled siRNA or siRNA for HIF1A and HIF2A, as indicated. (c) Lysates of TB cells treated as in (a and b) (key on right) were assayed by western blot for HSPA6 (70 kDa) and β-actin (45 kDa). MMP2 (d) and HBEGF (e) ELISAs with lysates of TB cells treated as in a and b.*, P<0.005, compared with control (Vehicle 20% O2), n=3

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Control

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Regulation of MMP2, HBEGF and cell survival by HSP70. HSP70 was inhibited using the pharmacological inhibitor VER 155008 during culture of HTR-8/SVneo cells for 4 h at 20% or 2% O2. At 2% O2, HSP70 inhibition caused a dose-dependent decrease in both MMP2 (a) and HBEGF (b), measured by immunohistochemistry, and concomitantly increased cell death (c), measured by TUNEL. Cells cultured at 20% O2 were unaffected. (d) ELISA for HBEGF in cellular extracts of TB cells cultured for 4 h at either 20% or 2% O2 in the presence of 10 nM recombinant MMP2 (rMMP2) and 5 μM HSP70 inhibitor (VER 155008), as indicated. *P<0.05, compared with vehicle/20% O2, n=3

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Inhibition, Immunohistochemistry, TUNEL Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Regulation of MMP2 in first trimester chorionic villous explants. First trimester villous explants cultured for 6 h at either 8% or 2% O2 in the presence of recombinant MMP2 (rMMP2) and VER 155008 (HSP70 inh.), as indicated, were stained for MMP2 & RT7 (a), HBEGF &RT7 (c) and counterstained with Hematoxylin (blue). Cell extracts were assayed by ELISA for MMP2 (b) and HBEGF (d) Sections of villous explants were dual stained with DAPI and TUNEL (e), using immunofluorescence microscopy and quantified for apoptosis (f). Size bars in a and c indicate 10 μm, and in (e) indicate 100 μm. *P<0.0001 in (b and d), P<0.05 in (f), compared with vehicle/8% O2, n=3

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Cell Culture, Recombinant, Staining, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Immunofluorescence, Microscopy

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Proximity ligation assay (PLA) for protein interactions during survival signaling. HTR-8/SVneo cells were cultured at 2% O2 for 4 h and duo labeled with non-immune IgG (a) or combinations of antibodies against HSPA6 (HSP70B′), MMP2 and HBEGF (b–d, as indicated). Cell nuclei were stained with DAPI (blue), and positive interactions between the indicated proteins in (b–d) are labeled red (PLA signal). Size bars indicate 50 μm

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Proximity Ligation Assay, Cell Culture, Labeling, Staining

Journal: Cell Death and Differentiation

Article Title: Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding

doi: 10.1038/cdd.2017.104

Figure Lengend Snippet: Proposed mechanism for regulation of HBEGF by O2. Low (2%) O2 upregulates HSP70 (1). HSP70 interacts with MMP2 (2) and regulates the accumulation of activated MMP2 at the cell surface (3), where it interacts with proHBEGF (4) possibly as its sheddase. The released sHBEGF binds (5) to its receptors, ERBB1 and ERBB4, through its EGF-like domain, and to heparan sulfate proteoglycans (HSPG) through its heparin-binding domain. ERBB downstream signaling activates MAPKs (6) required for biosynthesis of proHBEGF from the pool of HBEGF mRNA (7). HBEGF autocrine signaling thereby increases HBEGF secretion to achieve extracellular HBEGF concentrations (>1 nM) sufficient to inhibit apoptosis at 2% O2. Red arrows indicated that a direct interaction was demonstrated using proximity ligation assays

Article Snippet: 56 Rehydrated sections of cultured explants were labeled for 1 h at 25 °C with 5 μ g/ml goat polyclonal antibody against MMP2 and human recombinant HBEGF (R&D Systems) that recognizes both membrane and secreted forms of the protein and 2.5 μ g/ml antibody against cytokeratin (CK7) specific for TB and counterstained with hematoxylin.

Techniques: Binding Assay, Ligation