anti ephrinb3  (R&D Systems)

Journal: PLoS ONE

Article Title: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly

doi: 10.1371/journal.pone.0067015

Figure Lengend Snippet: (A) Representative images of growth cones from control and RhoA cKO cortical neurons after a 30 min incubation with preclustered ephrinB3-Fc or Fc alone. Scale bar:10 µm. (B) Quantification of the growth cone collapse response of control and RhoA cKO neurons treated with ephrinB3-Fc or Fc (*p<0.001, ANOVA followed by the Tukey test). (C) Cortical neurons from RhoA cKO mice express EphA4 at levels similar to control neurons. (D) Percentage of ephrinB3-induced growth cone collapse in rat cortical neurons pretreated or not with the ROCK inhibitor Y-27632 (*p<0.001, ANOVA followed by the Tukey test). (E) Expression of ephrinB3 is lost from the midline at L2 in RhoA cKO mice. Arrowheads show ephrinB3 positive staining in the spinal cord of P5 mice. Scale bar: 10 µm. Three sections per animal and N = 3 animals were analyzed per genotype.

Article Snippet: The following purchased antibodies were used in our study: rabbit polyclonal antibodies anti-RhoA, anti-RhoB, and anti-RhoC (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-EphA4 receptor and rabbit polyclonal anti-ephrinB3 (Invitrogen, Camarillo, CA); anti-GAPDH antibody (Millipore, Temecula, CA); and the mouse monoclonal anti-acetylated tubulin antibody (Sigma, St. Louis, MO).

Techniques: Incubation, Expressing, Staining

Journal: PLoS ONE

Article Title: The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly

doi: 10.1371/journal.pone.0067015

Figure Lengend Snippet: In control mice, when EphA4-expressing CST and spinal interneuron (IN) axons encounter ephrinB3 at the spinal cord midline, they are repulsed due to the initiation of signaling pathways that involve RhoA activation and Rac1 inhibition. Loss of RhoA causes aberrant midline crossing of CST and spinal IN axons due to a failure of neurons to retract their axons and/or the absence of ephrinB3 expression at the midline.

Article Snippet: The following purchased antibodies were used in our study: rabbit polyclonal antibodies anti-RhoA, anti-RhoB, and anti-RhoC (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-EphA4 receptor and rabbit polyclonal anti-ephrinB3 (Invitrogen, Camarillo, CA); anti-GAPDH antibody (Millipore, Temecula, CA); and the mouse monoclonal anti-acetylated tubulin antibody (Sigma, St. Louis, MO).

Techniques: Expressing, Activation Assay, Inhibition

Journal: Acta neuropathologica

Article Title: Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling.

doi: 10.1007/s00401-019-02011-1

Figure Lengend Snippet: Fig. 6 EphrinB3 improves SC migration on FN-coated surfaces. a, b Exit of SC entrapped in an agarose drop and seeded on FN + Fc (a) and FN + EphrinB3 (b), scale bar 200 µm. (c) More SC exit the agarose drop 5 h post-seeding on FN + EphrinB3 than on con- trol surface (two-way ANOVA with repeated measures: p = 0.03, F(1,4) = 10.24). d SC migrate over longer distances from the drop-edge 4 h post- seeding (two-way ANOVA with repeated measures: p = 0.02, F(1,4) = 12.74) on FN + EphrinB3-coated surfaces (n = 3 per group). Graphs represent the values of separate experiments (mean ± SD). e Velocity of single SC was measured in different condi- tions. Only SC seeded on FN + EphrinB3 show a sig- nificant increase of migration speed. This increase is reverted when SC are pre-incubated with anti-EphA4 (one-way ANOVA p = 0.002, F(5,93) = 4.1; two-tailed Mann–Whitney test p = 0.002 between control FN + Fc and FN + EphrinB3 group, n = 12–18 per group). In e, n represents tracked single cells of three different experi- ments repeated independently. Data are expressed as mean values ± SD. Single cells were tracked from three different independent experiments, **p < 0.01

Article Snippet: Neutralization of EphrinB3 epitopes in myelin extracts EphrinB3 epitopes in myelin extract proteins were neutralized by incubation with anti-EphrinB3 antibodies (AF395, R&D system and sc-271328, Santa Cruz Biotechnology, ratio: 1:1) for 2 h at room temperature prior to the addition to the cells [55].

Techniques: Migration, Incubation, Two Tailed Test, MANN-WHITNEY, Control

Journal: Acta neuropathologica

Article Title: Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling.

doi: 10.1007/s00401-019-02011-1

Figure Lengend Snippet: Fig. 8 Model of the mechanism of guidance and migration of SC after CNS demyelination. a, b SC encountering CNS white matter are activated by the myelin-associated EphrinB3 through EphB6- and EphA4-SC receptors. c, d The activation of these receptors by phospho- rylation impairs SC adhesion to white matter and increases SC expression of Integrinβ1, promoting their adhesion to BV extracellular matrix. e Lesions of white matter undergo the formation and/or remodeling of BV which increases expression of ECM adhesion molecules, such as FN, and further facili- tate SC mobilization towards the lesion

Article Snippet: Neutralization of EphrinB3 epitopes in myelin extracts EphrinB3 epitopes in myelin extract proteins were neutralized by incubation with anti-EphrinB3 antibodies (AF395, R&D system and sc-271328, Santa Cruz Biotechnology, ratio: 1:1) for 2 h at room temperature prior to the addition to the cells [55].

Techniques: Migration, Activation Assay, Expressing