Journal: The Journal of Neuroscience

Article Title: Specification of Optic Nerve Oligodendrocyte Precursors by Retinal Ganglion Cell Axons

doi: 10.1523/JNEUROSCI.0855-06.2006

Figure Lengend Snippet: Retinal induction of OPCs is dependent on hedgehog signaling. A–F, Stage 24 (E4) retinal explant conditioned medium induces OPCs in stage 24 (E4) dorsal spinal cord cells after 4 d. A, B, Control cultures containing few OPCs. C, D, Retinal CM induces significant numbers of OPCs. E, F, This induction is inhibited by treatment with cyclopamine. G, Quantification of O4+ cells in cultures treated with Shh function-blocking mAb 5E1 shows that the induction is inhibited in a dose-dependent manner. Error bars represent SD (3 different preparations). H, I, In cocultures of retinal and dorsal spinal cord explants, the induction of OPCs is reduced significantly by cyclopamine treatment. J, Quantification of O4+ cells in control cocultures and cocultures treated with cyclopamine. K, L, Likewise, the inductive capacity of retinal neurites in the Campenot chambers is blocked by the addition of cyclopamine to the lateral chamber. M, Quantification of O4+ cells in Campenot chambers. Scale bars (in D, K), 50 μm.

Article Snippet: 5E1 antibody (Developmental Studies Hybridoma Bank) and anti-NRG antibody were added to the culture.

Techniques: Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Specification of Optic Nerve Oligodendrocyte Precursors by Retinal Ganglion Cell Axons

doi: 10.1523/JNEUROSCI.0855-06.2006

Figure Lengend Snippet: Appearance of OPCs at the midline of the third ventricle is blocked by cyclopamine treatment, and localized Shh expression is absent in eyeless animals. A, B, Exposure to cyclopamine between stage 21 (E3) and stage 29 (E6) inhibited the normal development of OPCs lining the third ventricle. C–H, Explants derived from the third ventricle floor developed reduced numbers of O4+ cells after cyclopamine exposure. Shown are control explants (C, D), explants from cyclopamine-injected animals (E, F), and explants exposed to cyclopamine in vitro (G, H). I, Quantification of OPCs in explants from animals exposed to cyclopamine. J, Quantification of OPCs in explants treated with cyclopamine after removal from the naive animals. The reduction in OPC number was greater in explants from animals exposed to cyclopamine (I) than in explants treated with cyclopamine after removal (J), suggesting that OPC induction occurred before explant establishment. K, Shh is detectable by mAb 5E1 staining in the chiasmal region (arrow) of the optic nerve only in the presence of optic nerve axons. L, In eyeless animals, the labeling is lost, although it is retained in lateral aspects of the third ventricle (arrowhead). M, Expression of patched is detected at the floor of the third ventricle. N, O, In wild-type embryos at stage 27 (E5) and stage 31 (E7) Shh mRNA is not expressed at the floor of the third ventricle adjacent to the optic chiasm, although it is expressed laterally (on, optic nerve). P, At stage 31 (E7) in eyeless embryos the pattern of Shh mRNA expression was not altered significantly. Scale bars: A, B, M, O, P, 100 μm; C–H, K, L, N, 50 μm.

Article Snippet: 5E1 antibody (Developmental Studies Hybridoma Bank) and anti-NRG antibody were added to the culture.

Techniques: Expressing, Derivative Assay, Injection, In Vitro, Staining, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Chimeric 5E1 is functionally equivalent to murine 5E1. A, schematic of murine 5E1 (m5E1, yellow) IgG (33) and its chimeric counterpart (ch5E1), where the constant domains (CH1–3 and CL) have been replaced with the corresponding domains from the humanized antibody trastuzumab (blue) (35), leaving the variable light and heavy (VL and VH) domains of the murine 5E1 antibody intact. B, ch5E1 and m5E1 bind similarly to an endogenous Hh-expressing cell line. Flow cytometry analysis is shown of endogenous Hh in HT29 cells with the indicated concentrations of m5E1 or ch5E1 (in μg/ml). The means ± S.D. of triplicate reactions are plotted. C, ch5E1 (■) and m5E1 (●) bind similarly to stably transfected Shh-COS cells by flow cytometry analysis. The means ± S.D. of a representative duplicate experiment are shown. Isotype controls (chimeric IgG (□) or murine IgG1 (○)) show no appreciable binding. D, ch5E1 and m5E1 compete for cell surface Shh. The ability of increasing amounts of ch5E1 (■) or m5E1 (●) to compete with ∼0.69 nm (0.1 μg/ml) m5E1 for binding to Shh-expressing cells and vice versa as monitored by flow cytometry analysis is shown, normalized to 100% for no competitor after background subtraction. Isotype controls (murine IgG1 (○) or chimeric IgG (□)) are unable to compete for Shh binding. E, ch5E1 specifically detects Hh in the developing mouse embryo. E10.5 embryos were sectioned and stained with m5E1 (top) or ch5E1 (bottom), followed by Cy3-conjugated secondary antibodies (left panel and red in merged right panel) and 4′,6-diamidino-2-phenylindole (DAPI) (blue nuclear staining in right merged panels). ch5E1 is as specific as m5E1 in detecting Hh in the notochord (NC) and floor plate (FP). Scale bar is 200 μm (images taken at ×10 magnification). F, ch5E1 and m5E1 inhibit Hh signaling similarly. HT29 cells secreting Hh were co-cultured with S12 cells (C3H10T1/2 cells stably expressing a Gli-luciferase reporter (36)). Hh signaling was stimulated by serum starvation for 24 h in the presence of the indicated concentrations of ch5E1 (■), m5E1 (●), hIgG1 (▴), or mIgG1 (○) antibodies. The means ± S.D. of the luciferase signals (RLU; relative luminescence units) of triplicate measurements are plotted. This experiment is representative of multiple independent experiments.

Article Snippet: After two washes in FACS buffer, the 5E1 signal was amplified with 1:100 biotinylated goat anti-mouse for m5E1 (catalog no. 115-065-071, Jackson ImmunoResearch) or anti-human for ch5E1 (catalog no. 109-065-003, Jackson ImmunoResearch) for 1 h prior to two washes and detection with 1:50 rhodamine streptavidin-phycoerythrin (catalog no. 016-110-084, Jackson ImmunoResearch).

Techniques: Expressing, Flow Cytometry, Stable Transfection, Transfection, Binding Assay, Staining, Cell Culture, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Binding kinetics of Hh ligands to ch5E1 Fab in the presence and absence of divalent ions by biolayer interferometry Binding measurements for human and rat Shh were carried out using immobilized Hh ligand and ch5E1 or m5E1 Fabs in solution. Binding measurements for human Ihh and human Dhh were carried out using immobilized ch5E1 and Hh ligand in solution as described under “Experimental Procedures.”

Article Snippet: After two washes in FACS buffer, the 5E1 signal was amplified with 1:100 biotinylated goat anti-mouse for m5E1 (catalog no. 115-065-071, Jackson ImmunoResearch) or anti-human for ch5E1 (catalog no. 109-065-003, Jackson ImmunoResearch) for 1 h prior to two washes and detection with 1:50 rhodamine streptavidin-phycoerythrin (catalog no. 016-110-084, Jackson ImmunoResearch).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: 5E1 binding to Shh is enhanced by divalent cations. Biolayer interferometry sensorgrams of ch5E1 Fab binding to C-terminally biotinylated Shh on streptavidin-coated biosensors are shown in the absence (A) or presence (B) of Ca2+ and Zn2+. Sensorgrams of seven 2-fold serial dilutions of ch5E1 Fab starting at 125 nm are shown, where 125 nm results in the largest response. C, isothermal titration calorimetry of human Shh with ch5E1 Fab. Experiments were carried out in the absence or presence of Ca2+ (5 μm CaCl2) and/or Zn2+ (500 μm ZnSO4) as indicated. As expected, the stoichiometry of 5E1-Shh complex formation in solution derived from the ITC data indicates 1:1 binding in all cases.

Article Snippet: After two washes in FACS buffer, the 5E1 signal was amplified with 1:100 biotinylated goat anti-mouse for m5E1 (catalog no. 115-065-071, Jackson ImmunoResearch) or anti-human for ch5E1 (catalog no. 109-065-003, Jackson ImmunoResearch) for 1 h prior to two washes and detection with 1:50 rhodamine streptavidin-phycoerythrin (catalog no. 016-110-084, Jackson ImmunoResearch).

Techniques: Binding Assay, Isothermal Titration Calorimetry, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

doi: 10.1074/jbc.M110.112284

Figure Lengend Snippet: Competition binding of ch5E1 Fab with Hhip L2 peptide binding to Shh. Biolayer interferometry sensorgrams of 250 nm Shh binding to biotinylated Hhip L2 peptide on streptavidin-coated biosensors are shown in the presence and absence of various concentrations of ch5E1 Fab. The inset shows the fraction response after binding at equilibrium versus the molar ratio of ch5E1 Fab/Shh. Because Shh and ch5E1 concentrations used are well above the KD value, ch5E1 effectively titrates Shh, having a molar ratio of 1.25 (x axis intercept), close to the predicted value of 1.0. At molar ratio 2.4, there is no observed binding of Shh to the Hhip L2 peptide.

Article Snippet: After two washes in FACS buffer, the 5E1 signal was amplified with 1:100 biotinylated goat anti-mouse for m5E1 (catalog no. 115-065-071, Jackson ImmunoResearch) or anti-human for ch5E1 (catalog no. 109-065-003, Jackson ImmunoResearch) for 1 h prior to two washes and detection with 1:50 rhodamine streptavidin-phycoerythrin (catalog no. 016-110-084, Jackson ImmunoResearch).

Techniques: Binding Assay