rabbit anti epha4 polyclonal antibody  (Santa Cruz Biotechnology)

Journal: Frontiers in Neurology

Article Title: Repetitive trans-spinal magnetic stimulation improves motor function in rats with spinal cord injury and is associated with upregulation of EphA4 signaling pathway proteins

doi: 10.3389/fneur.2026.1726570

Figure Lengend Snippet: Relative expression level of EphA4 mRNA detected by RT–PCR. rTSMS treatment effectively increased EphA4 mRNA expression in rats with acute SCI, with this effect potentially peaking at approximately week 4. The data are presented as the means ± standard deviations. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. CON, control group; rTSMS, repetitive trans-spinal magnetic stimulation group; S-rTSMS, sham stimulation group; SO, sham-operated.

Article Snippet: For immunofluorescence, the following primary antibodies were used: a rabbit polyclonal anti-EphA4 antibody (Product # 21875-1-AP, Proteintech; 1:50 dilution), which targets a protein of approximately 120 kDa, and a rabbit monoclonal anti-VGluT2 antibody (Product # DF13296, Affinity Biosciences; 1:200 dilution), targeting a protein of about 64 kDa.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Frontiers in Neurology

Article Title: Repetitive trans-spinal magnetic stimulation improves motor function in rats with spinal cord injury and is associated with upregulation of EphA4 signaling pathway proteins

doi: 10.3389/fneur.2026.1726570

Figure Lengend Snippet: Protein levels of EphA4, EphrinB3, and the downstream proteins Chn1 and Nck1. rTSMS treatment effectively promoted the synthesis of EphA4, EphrinB3, Chn1, and Nck1 in rats with acute SCI. (A) Representative western blot images of EphA4, EphrinB3, Chn1, and Nck1 at weeks 2, 4, and 6. (B) Relative expression level of EphA4 at each time point ( n = 3). (C) Relative expression level of EphrinB3 at each time point ( n = 3). (D) Relative expression level of Chn1 at each time point ( n = 3). (E) Relative expression level of Nck1 at each time point ( n = 3). The data are presented as the means ± standard deviations. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. CON, control group; rTSMS, repetitive transspinal magnetic stimulation group; S-rTSMS, sham stimulation group; SO, sham-operated group.

Article Snippet: For immunofluorescence, the following primary antibodies were used: a rabbit polyclonal anti-EphA4 antibody (Product # 21875-1-AP, Proteintech; 1:50 dilution), which targets a protein of approximately 120 kDa, and a rabbit monoclonal anti-VGluT2 antibody (Product # DF13296, Affinity Biosciences; 1:200 dilution), targeting a protein of about 64 kDa.

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Neurology

Article Title: Repetitive trans-spinal magnetic stimulation improves motor function in rats with spinal cord injury and is associated with upregulation of EphA4 signaling pathway proteins

doi: 10.3389/fneur.2026.1726570

Figure Lengend Snippet: Coexpression of EphA4 and VGluT2 detected by immunofluorescence. Six weeks of rTSMS treatment effectively promoted the synthesis of EphA4 and VGluT2 in cells of the ventral horn of the spinal cord in rats with acute SCI. Panels (A,F,K,P) show low-magnification immunofluorescence overviews of the spinal cord. The ventral horn cell region is outlined by a purple dashed line. A red dashed line indicates the dorsoventral axis. The yellow rectangle marks the specific area selected for high-magnification imaging in the subsequent panels. The scale bar represents 200 μm. High-magnification views (Panels B–E , G–J , L–O , Q–T ) are displayed at a uniform scale of 20 μm (scale bars shown). In these panels, immunopositive signals for EphA4 (detected with Cy3) appear in red, while those for VGluT2 (detected with EGFP) appear in green. Co-localization of EphA4 and VGluT2 immunoreactivity results in a yellowish signal. (U) Proportion of EphA4/VGluT2 double-labeled immunopositive neurons relative to the total cell count ( n = 3). (V) Proportion of EphA4-labeled neurons relative to the total cell count ( n = 3). (W) Proportion of VGluT2-labeled neurons relative to the total cell count ( n = 3). The data are presented as the means ± standard deviations. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. CON, control group; rTSMS, repetitive transspinal magnetic stimulation group; S-rTSMS, sham stimulation group; SO, sham-operated group.

Article Snippet: For immunofluorescence, the following primary antibodies were used: a rabbit polyclonal anti-EphA4 antibody (Product # 21875-1-AP, Proteintech; 1:50 dilution), which targets a protein of approximately 120 kDa, and a rabbit monoclonal anti-VGluT2 antibody (Product # DF13296, Affinity Biosciences; 1:200 dilution), targeting a protein of about 64 kDa.

Techniques: Immunofluorescence, Imaging, Labeling, Cell Characterization, Control

Journal: bioRxiv

Article Title: Plasmin-mediated cleavage of EphA4 at central amygdala inhibitory synapses controls anxiety

doi: 10.1101/2021.07.16.452595

Figure Lengend Snippet: (a) Treatment of SHSY-5Y cells with increasing concentrations of plasmin (14 nM of tPA and 6, 12 or 120 nM of plasminogen, shown as +, ++ or +++, respectively) reduced the density and eventually caused disappearance of the EphA4 band in Western blotting, consistent with plasmin cleaving EphA4 (F ( , ) = 14.6; p<0.05 and p<0.001). Plasmin cleavage of EphA4 was specific because other Eph receptors remained intact (Supplementary Fig. 2). (b) Addition of increasing concentrations of plasmin (as above) to the mouse amygdala homogenate caused a decrease in the density of the native EphA4 band and concomitant appearance of a novel ~50 kDa EphA4 band, demonstrating the capacity of plasmin to cleave EphA4 in the brain milieu. EphA4 co-localise with tPA and plasminogen in gephyrin positive synapses (c) in the central amygdala, scale bar is 100nm. (d) Mice were subjected to restraint stress, their amygdalae dissected and EphA4 visualized by Western blotting. Two-fold increase in the density of EphA4 cleavage band was observed in stressed wild-type (F ( , ) = 8.7, p< 0.01, WT stress vs WT no-stress), but not in stressed tPA −/− or Plg −/− animals (F ( , ) = 0.6 and F ( , ) = 1.9 respectively, p>0.05). Representative Western blots for tPA −/− and Plg −/− animals are shown in Supplementary Fig. 3 (e) Treatment of the purified recombinant EphA4-Fc protein with increasing concentrations of plasmin (as in a ) caused a decrease in the density of the main EphA4 band and a concomitant appearance of a novel ~50 kDa EphA4 band in the Western blotting, demonstrating that plasmin cleaves EphA4 directly. Similar results were obtained when anti-EphA4 N-terminal (instead of C-terminal) antibodies were used (Supplementary Fig. 3 f). Results are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: Homogenates were incubated for 1 hour with 2 μg (1:250) of either an irrelevant IgG from the same species (Cell Signalling Technology, USA) or rabbit anti-EphA4 polyclonal antibody (Proteintech, UK).

Techniques: Western Blot, Purification, Recombinant

Journal: bioRxiv

Article Title: Plasmin-mediated cleavage of EphA4 at central amygdala inhibitory synapses controls anxiety

doi: 10.1101/2021.07.16.452595

Figure Lengend Snippet: (a) tPA and plasmin cleavage sites of EphA4 were identified by amino-terminal oriented mass spectrometry of substrates (ATOMS) after treating purified recombinant EphA4-Fc with either tPA or tPA + increasing concentrations of plasminogen (see Methods). Diagram shows the sensitivity and localisation of the cleavage sites relative to the structure of EphA4. Plasmin cleaved EphA4 with 100-fold higher efficiency than tPA. (b) The cleavage site P5, located within the fibronectin type-III domain of EphA4, was the most sensitive to cleavage by plasmin. Cleavage at P5 is consistent with the size of the EphA4 plasmin cleavage bands shown in . (c) Co-immunoprecipitation followed by Western blotting revealed that in the amygdala EphA4 physically interacts with the neuro-skeleton protein gephyrin, a universal regulator of GABA-receptor subunit anchoring. (d, e) Further co-immunoprecipitation studies demonstrated that EphA4/gephyrin interaction is dynamic and regulated by plasmin cleavage of EphA4 at R497. EphA4/gephyrin binding is enhanced upon the expression of plasmin-resistant variant of EphA4 (prEphA4), and disrupted upon the expression of EphA4 truncated at R497 (tEphA4) (F ( , ) = 42.64, P=0.0003, **P<0.01)

Article Snippet: Homogenates were incubated for 1 hour with 2 μg (1:250) of either an irrelevant IgG from the same species (Cell Signalling Technology, USA) or rabbit anti-EphA4 polyclonal antibody (Proteintech, UK).

Techniques: Mass Spectrometry, Purification, Recombinant, Immunoprecipitation, Western Blot, Binding Assay, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Plasmin-mediated cleavage of EphA4 at central amygdala inhibitory synapses controls anxiety

doi: 10.1101/2021.07.16.452595

Figure Lengend Snippet: (a) Examples of primary mouse amygdala neurons over-expressing wtEphA4, prEphA4 and tEphA4 variants, scale bar is 3µm. ( b ) Over-expression of prEphA4 favours formation of short dendritic spines with wide heads, while tEphA4 promotes generation of long spines with thin heads (for length/width ratio H(1) = 97.09, P<0.0001) (c) Comparison of dendritic spine length. Over-expression of tEphA4 in mouse amygdala neurons resulted in formation of longer spines compared to those transfected with EGFP-containing control vector (H(1) =20.64, P<0.0001) (d) Comparison of spine head width. Over-expression of prEphA4 resulted in formation of spines with wide heads, while overexpression of tEphA4 promoted formation of spines with narrow heads (H(1) = 125.3, P<0.0001). (e) Comparison of dendritic spine area. Over-expression of either wtEphA4 or prEphA4 led to a formation of larger spines, while overexpression of tEphA4 was devoid of such an effect (H(1) = 33.87, P<0.0001). (f) Dendritic spine density was not affected by the overexpression of EphA4 variants (F (3, 105) =0.68, P>0.05). The total number of spines analyzed was 1131 (13 neurons) for wtEphA4, 1088 (14 neurons) for prEphA4, 1173 (15 neurons) for tEphA4 and 1241 (9 neurons) for a control construct (containing EGFP only). Results are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: Homogenates were incubated for 1 hour with 2 μg (1:250) of either an irrelevant IgG from the same species (Cell Signalling Technology, USA) or rabbit anti-EphA4 polyclonal antibody (Proteintech, UK).

Techniques: Expressing, Over Expression, Comparison, Transfection, Control, Plasmid Preparation, Construct

Journal: bioRxiv

Article Title: Plasmin-mediated cleavage of EphA4 at central amygdala inhibitory synapses controls anxiety

doi: 10.1101/2021.07.16.452595

Figure Lengend Snippet: (a) UbC-EGFP-P2A-EphA lentiviral vectors, containing either wtEphA4, prEphA4 or tEphA4 (or EGFP as control), were bilaterally injected into the central amygdala (CEA) and mice were allowed to recover for three weeks to allow the transgene expression. Then, mice were subjected to restraint stress (or left undisturbed) and their anxiety levels measured in the elevated-plus maze. Representative injection sites, identified by the expression of EGFP, are shown in the middle panel. (b) In UbC-EGFP-injected mice, restraint stress caused a decrease in the time spent in open arms, indicative of elevated levels of anxiety. Overexpression of either wtEphA4 or prEphA4 in the same vector prevented the development of stress-induced anxiety, supporting the view that uncleaved EphA4 promotes anxiolysis. Consistently, overexpression of tEphA4 in CEA dramatically enhanced stress-induced anxiety, altogether demonstrating that plasmin cleavage of EphA4 at R497 is necessary for this process (F (7, 65) = 6.796; P<0.0001; **p<0.01, ***p<0.01). (c) Representative elevated-plus maze traces of mice subjected to restraint stress. Results are shown as mean ± SEM. **p<0.01, ***p<0.001

Article Snippet: Homogenates were incubated for 1 hour with 2 μg (1:250) of either an irrelevant IgG from the same species (Cell Signalling Technology, USA) or rabbit anti-EphA4 polyclonal antibody (Proteintech, UK).

Techniques: Control, Injection, Expressing, Over Expression, Plasmid Preparation

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: (A) Schemes of wild-type (WT) and a cytoplasmic deletion mutant of EphA4. The numbers represent the amino acid numbers counting from the one encoded by the initiation codon. TM, transmembrane domain; JM, juxtamembrane domain. Dotted blue rectangles indicate the deleted amino-acid regions. (B) Binding between the extracellular domains of EphA4 and GHR. EphA4(WT)-3Flag or EphA4(Δcyto)-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells. These proteins were subjected to co-immunoprecipitation (IP), fractionation with SDS-PAGE, and detection by immunoblotting (IB) using the indicated antibodies. Arrowheads indicate the molecules shown in the figure. The other bands are regarded as non-specific bands, compared with the result without EphA4 expression (see also ). (C) Binding between the cytoplasmic domains of EphA4 and GHR. GHR(WT)-6myc and the chimeric protein in which the cytoplasmic domain of EphA4 was fused to the extracellular domain of ephrin-B2 (B2-A4-3Flag) were co-expressed in HEK293T cells and their interaction was examined by IB following IP and SDS-PAGE fractionation. The bands at approximately 100 kDa for GHR-6myc transfectants probably correspond to the GHR molecules modified with carbohydrates. (D) Control experiment for (C) showing no interaction between GHR and ephrin-B2. Ephrin-B2-3Flag was transiently co-expressed with GHR(WT)-6myc in HEK293T cells and their interaction was examined as described in (C). Some of the molecules that have extracellular domains (EphA4, ephrin-B2, and B2-A4) show broad bands, with the upper bands represented by the carbohydrate-modified proteins. All the IP and IB studies in this figure were repeated 3 times to confirm reproducibility, and representative results are shown.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, Fractionation, SDS Page, Western Blot, Expressing, Modification

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: (A) Schemes of WT and various cytoplasmic mutants of EphA4. The numbers represent the amino acid numbers counting from the one encoded by the initiation codon. TM, transmembrane domain; JM, juxtamembrane domain. Dotted blue rectangles indicate the deleted amino-acid regions. (B) Binding between EphA4 mutants and JAK2. EphA4(WT) and mutants, which were tagged with 3Flag, were transiently co-expressed with JAK2(WT)-6myc in HEK293T cells. Cell lysates were used for IP and/or IB studies with the indicated antibodies. Transfection with DNA for vector alone (vector) in place of DNA for EphA4 mutants was used as a control. Cells that were treated for transfection with no DNA (No DNA transfected) were used to exclude contamination of any transfected DNA with other DNA, showing that plasmid DNA for vector or JAK2-6myc does not change the IP and IB results shown in the figure. Arrowheads indicate the molecules expressed after transfection. The other bands are regarded as non-specific bands in comparison with the result of vector transfection (see also ). The same experiments were repeated 3 times, showing similar results. (C) Binding between EphA4 mutants and STAT5B. EphA4(WT) and mutants, which were tagged with 3Flag, were transiently co-expressed with STAT5B(WT)-6myc in HEK293T cells. Cell lysates were used for IP and/or IB studies with the indicated antibodies. The lines drawn between the lanes are to indicate that a lane was omitted due to a technical error in loading samples on the gel. All samples were fractionated by SDS-PAGE at the same time and immunoblotted on the same membrane. The same experiments were repeated twice, showing essentially the same results.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: Binding Assay, Transfection, Plasmid Preparation, SDS Page

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: (A) Schemes to show JAK2(WT) and its deletion mutants. Dotted blue rectangles indicate the deleted amino-acid regions. (B) JAK2(WT) and mutants, which were tagged with 3Flag, were transiently co-expressed with EphA4(WT)-HA in HEK293T cells, and the cells were exposed to 0.5 μg/mL oligomerized ephrin-A1 for 75 min. Cell lysates were used for IP and/or IB studies with the indicated antibodies. All samples were fractionated by SDS-PAGE at the same time and immunoblotted on the same membrane, except that 1 lane between JAK2(Δ1–240, Δ251–473) and JAK2(511) was omitted due to a technical error. Arrowheads indicate the JAK2 mutants expressed in the cells. The other bands are regarded as non-specific bands in comparison with the result of control vector transfection (see also ). The same experiments were repeated twice, showing similar results. Deletion of the carboxy-terminal half of the JAK2 molecule abolished the binding of JAK2 to EphA4.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: SDS Page, Plasmid Preparation, Transfection, Binding Assay

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: (A) GHR phosphorylation by EphA4 in the absence of JAK2. Jak2 -KO mouse embryonic fibroblasts (MEF) retrovirally overexpressing EphA4 (EphA4 overexp) and those overexpressing EphA4 and Ghr (EphA4&GHR overexp) were pre-incubated with serum-free medium overnight and incubated with ephrin-A1 (0.5 μg/mL) for the time shown. Cell lysates were used for IP and/or IB studies with the indicated antibodies. Anti-pY: anti-phosphotyrosine antibody. The same experiments were repeated 3 times, showing the same results. (B) JAK2 phosphorylation by EphA4. HEK293T cells expressing JAK2(KD)-Flag with or without EphA4(WT)-HA were preincubated with serum-free medium for 6 h, then stimulated with ephrin-A1 (0.5 μg/mL) for the time shown. Cell lysates were detected by IB and/or IP with the indicated antibodies. The same experiments were repeated 3 times, showing the same results.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: Incubation, Expressing

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: (A) Structure of GHR WT and mutants. The numbers represent the amino acid numbers counting from the initiation codon. “Box1,” which is the proline-rich region of the cytoplasmic domain, is necessary for GH-dependent association with JAK2 and the following activation of JAK2 . TM: transmembrane domain. The numbers represent the amino acid numbers counting from the one encoded by the initiation codon. (B) Binding of JAK2 with GHR (WT and mutants) and phosphorylation of STAT5B. GHR mutants tagged with HA were co-expressed with JAK2-myc in HEK293T cells and the cells were stimulated with GH for 15 min. GHR(464/2F) bound to and activated JAK2, but did not phosphorylate STAT5B. (C) Phosphorylation of STAT5B bound to kinase-deleted EphA4 by GH-activated JAK2. GHR(WT), (464/2F), or (Δcyto) was co-expressed in HEK293T cells with EphA4(ΔJM,ΔK)-3Flag, which binds to STAT5B , then the cells were stimulated with GH for 15 min. The expression of EphA4(ΔJM,ΔK)-3Flag did not alter the extent of STAT5B phosphorylation as compared with the control (vector). The cell lysates were used for IP and/or IB with the indicated antibodies. Arrowheads indicate the molecules expressed following transfection. The other bands are regarded as non-specific bands in comparison with the result of vector transfection (see also ). GHR molecules are detected as double or diffuse bands with the upper bands representing carbohydrate-modified molecules. All the experiments in (B) and (C) were repeated twice, showing similar results.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: Activation Assay, Binding Assay, Expressing, Plasmid Preparation, Transfection, Modification

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: (A) EphA4-HA was expressed in HEK293T cells, then stimulated with oligomerized ephrin-A1-Fc, ephrin-A5-Fc, ephrin-B2-Fc, or IgG-Fc alone as shown in the Materials and Methods for the time indicated. Cell lysates were fractionated with SDS-PAGE. pSTAT5B was detected by IB following IP using the antibodies shown, and EphA4-HA was detected with an anti-HA antibody. The same experiments were repeated 3 times, showing similar results.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: SDS Page

Journal: PLoS ONE

Article Title: Molecular interactions of EphA4, growth hormone receptor, Janus kinase 2, and signal transducer and activator of transcription 5B

doi: 10.1371/journal.pone.0180785

Figure Lengend Snippet: Interactive binding of the molecules participating in the GH/GHR and ephrin/Eph signal transduction pathways is shown in the left-side panel. When both GH and ephrin were applied to the cells, GHR and EphA4 bind to each other through both extracellular and intracellular domains. Then, JAK2 and STAT5B are attracted to the complex: the amino-terminal region of JAK2 to GHR, the carboxy-terminal region to EphA4, and STAT5B to the EphA4 kinase domain in addition to the known binding of STAT5B to the cytoplasmic domain of GHR. The middle panel shows the actions of JAK2 kinase and EphA4 kinase in response to GH. The activated complex-bound JAK2 phosphorylates both EphA4 and STAT5B that are bound to GHR, but not STAT5B bound to EphA4. EphA4 kinase activity is also enhanced by JAK2-mediated phosphorylation leading to the enhanced phosphorylation of STAT5B that is bound to EphA4. The right panel indicates the actions of EphA4 kinase and JAK2 kinase in response to ephrin. The activated EphA4 kinase phosphorylates JAK2, GHR, and STAT5B bound to EphA4. The activated JAK2 is supposed to activate STAT5B bound to GHR subsequently. This ephrin-initiated signaling requires the presence of GHR. The molecular explanation for this requirement is unknown. P, phosphorylation; N, amino-terminal domain; C, carboxy-terminal domain; CM, cell membrane; ECS, extracellular space.

Article Snippet: The following antibodies were used in the current study: mouse anti-myc monoclonal (clone 9E10; Santa Cruz Biotechnology; catalog #sc-40); mouse anti-Flag antibody derived from hybridoma (Sigma-Aldrich Co.; FLAG M2; Catalog #F1804); mouse anti-HA monoclonal (Santa Cruz Biotechnology; catalog #sc-7392, marked as anti-HA(m)); rat monoclonal anti-HA high-affinity (Sigma-Aldrich; clone 3F10; catalog #11867423001, marked as anti-HA); rabbit anti-EphA4 polyclonal (Santa Cruz Biotechnology; catalog #sc-921); rabbit anti-GAPDH (Santa Cruz Biotechnology; catalog #sc-25778); mouse anti-phosphotyrosine (anti-pY) monoclonal (Upstate Biotechnology; clone 4G10; catalog #05–321); goat anti-mouse GHR polyclonal (R&D systems; catalog #AF1360); rabbit anti-JAK2 monoclonal (Cell Signaling Technology; catalog #3230); rabbit anti-phospho-JAK2 (Tyr1007/1008) (anti-pJAK2) polyclonal (Cell Signaling Technology; catalog #3771); mouse anti-STAT5B monoclonal (Santa Cruz Biotechnology; catalog #sc-1656); and rabbit anti-phospho-STAT5 (Tyr694) (anti-pSTAT5) polyclonal (Cell Signaling Technology; catalog #9351).

Techniques: Binding Assay, Transduction, Activity Assay

Journal: PLoS ONE

Article Title: Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α

doi: 10.1371/journal.pone.0128826

Figure Lengend Snippet: (A) Expression of all Eph receptors, ephrin ligands, FGFRs and related molecules in mouse embryonic NSPCs. RT-PCR was performed with equal amounts of total RNA isolated from mouse NSPCs. Fragment lengths are indicated on the left in base pairs. (B) Co-expression of FGFRs, EphA4 and FRS2α (green, left panel), respectively, with the neural stem cell marker nestin (red, middle panel) in cultured neurospheres. Merged images are shown in right panels. Neurospheres were cultured on PLL-coated plates for a short time, fixed and immunostained. Immunofluorescent images were detected using a confocal microscopy with an appropriate optical filter. (C, D) Inhibition of FGFR-EphA binding with a dominant-negative EphA4 molecule, EphA4(ΔJM,KD), tagged with enhanced green fluorescence protein (ΔJM,KD-EGFP). FGFR1-HA (C) and FGFR3-HA (D) were co-expressed with Flag-tagged EphAs (EphA1, 2, 3, 5 and 7), respectively, and increasing doses of ΔJM,KD-EGFP in HEK293T cells. Binding of FGFR-HA with EphAs-Flag was examined with immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting (IB) using the antibodies shown.

Article Snippet: The following antibodies were used according to the manufacturers’ instructions: mouse anti-myc antibody derived from hybridoma MYC1-9E10.2 (ATCC, Manassas, VA, USA; Cat. #CRL-1729), mouse anti-HA antibody derived from hybridoma 12CA5 (Roche, Mannheim, Germany; Cat. #11583816001), mouse anti-Flag M2 monoclonal antibody (clone M2; Sigma-Aldrich Co.; Cat. #F3165), mouse anti-HA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. #sc-7392), rat anti-HA monoclonal antibody (clone 3F10; Roche, Cat. #11867423001), mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology, Cat. #sc-9996); rabbit anti-FGFR1 polyclonal antibody (Santa Cruz, Cat. #sc-121), rabbit anti-FGFR3 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-123), rabbit anti-FRS2α polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8318), rabbit anti-EphA4 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-921), rabbit anti-Akt 1/2 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8312), rabbit anti-phospho-Akt (Ser473) monoclonal antibody (Cell Signaling Technology, Denvers, MA, USA; Cat. #4060), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cell Signaling Technology, Cat. #9101), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cell Signaling Technology, Cat. #9102), mouse anti-phosphotyrosine monoclonal antibody (clone 4G10, Millipore, Billerica, MA, USA Cat. #05–321) and rat anti-nestin antibody (Rat-401, DSHB, Iowa City, IA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Marker, Cell Culture, Confocal Microscopy, Inhibition, Binding Assay, Dominant Negative Mutation, Fluorescence, Immunoprecipitation, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α

doi: 10.1371/journal.pone.0128826

Figure Lengend Snippet: (A) Tyrosine phosphorylation of FRS2α and MAP kinase in response to FGF2 stimulation with and without ephrin-A1 pre-treatment. Mouse NSPCs were treated with FGF2 (10 ng/ml) for the indicated periods of time with or without 45-min ephrin-A1 (0.5 μg/ml) pre-treatment (PreA1). FRS2α was immunoprecipitated using anti-FRS2α Ab from the cell lysate. Immunoprecipitates were fractionated by SDS-PAGE, and phosphorylated FRS2α was detected by immunoblotting with anti-pY Ab. For MAP kinase, cell lysates (50 μg of total protein per time point) were fractionated by SDS-PAGE and immunoblotted with anti-phospho-p44/42 MAPK (Thr202/Tyr204) Ab (phospho-MAPK) and anti-p44/42 MAPK Ab (total MAPK). (B) Synergistic effect of ephrin-A1 and FGF2 on neurosphere formation. Single NSPCs were plated 500 cells/well in 96-well plates for 3–5 days in medium containing different concentrations and combinations of reagent. Reagents were added repeatedly on the 3rd and 5th days and the number of spheres counted on the 10th day under a microscope. Concentrations of ephrin-A1 are expressed in μg/ml and that of FGF2 in ng/ml. Values were analyzed using Tukey’s multiple comparison test following ANOVA; n = 10 for each value. Bars represent the SD. * p<0.01 compared to the control treated with IgG(Fc) alone or between two treatment groups. (C) Self-renewing activity of NSPCs expressing EphA4(WT), EphA4(DN), FRS2(WT) and FRS2(DN), respectively. Study protocols were as in (B), but cells were exposed to vehicle (black), ephrin-A1 (0.5 μg/ml; blue), FGF2 (20 μg/ml; yellow), or a combination of ephrin-A1 and FGF2 (green). Bottom panels show micrographs of the spheres at the same magnification after various treatments of vector-expressing cells. Values were analyzed using Tukey’s multiple comparison test following ANOVA; n = 4 for each value. Bars represent the SD. * p<0.01 compared to controls transduced with the vector alone and treated with the same reagent. Values between WT- and DN-expressing cells were also significantly different (p<0.01) for each treatment except those treated with vehicle alone.

Article Snippet: The following antibodies were used according to the manufacturers’ instructions: mouse anti-myc antibody derived from hybridoma MYC1-9E10.2 (ATCC, Manassas, VA, USA; Cat. #CRL-1729), mouse anti-HA antibody derived from hybridoma 12CA5 (Roche, Mannheim, Germany; Cat. #11583816001), mouse anti-Flag M2 monoclonal antibody (clone M2; Sigma-Aldrich Co.; Cat. #F3165), mouse anti-HA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. #sc-7392), rat anti-HA monoclonal antibody (clone 3F10; Roche, Cat. #11867423001), mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology, Cat. #sc-9996); rabbit anti-FGFR1 polyclonal antibody (Santa Cruz, Cat. #sc-121), rabbit anti-FGFR3 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-123), rabbit anti-FRS2α polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8318), rabbit anti-EphA4 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-921), rabbit anti-Akt 1/2 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8312), rabbit anti-phospho-Akt (Ser473) monoclonal antibody (Cell Signaling Technology, Denvers, MA, USA; Cat. #4060), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cell Signaling Technology, Cat. #9101), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cell Signaling Technology, Cat. #9102), mouse anti-phosphotyrosine monoclonal antibody (clone 4G10, Millipore, Billerica, MA, USA Cat. #05–321) and rat anti-nestin antibody (Rat-401, DSHB, Iowa City, IA, USA).

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Western Blot, Microscopy, Comparison, Control, Activity Assay, Expressing, Plasmid Preparation, Transduction

Journal: PLoS ONE

Article Title: Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α

doi: 10.1371/journal.pone.0128826

Figure Lengend Snippet: (A) Schematic representation of FRS2α mutants ectopically expressed in HEK293T cells. Mutant 4F contains phenylalanine in place of four tyrosine residues (Y169, Y306, Y349 and Y392), and is devoid of Grb2 binding. Similarly, mutant 2F contains phenylalanine in place of two tyrosine residues (Y439 and Y471), and is devoid of Shp2 binding. Mutant 6F carries the mutations of both 4F and 2F. (B, C) Tyrosine phosphorylation of FRS2α via FGFR1 and EphA4, respectively. Increasing doses (0, 1 and 3 μg per 60-mm plate) of pcDNA3.1/FGFR1-Myc or pcDNA3.1/EphA4-3Flag were co-transfected with pcDNA3.1/FRS2α (WT or mutants, 4 μg/plate) into HEK293T cells. A dominant-negative EphA4, EphA4(ΔJM,KD)-HA, was ectopically expressed in some HEK293T cells using a retrovirus vector (B, bottom panel). Some transfected cells were pretreated with 25 μM of SU5402 for 1 h before exposure to ephrin-A1 (C, bottom). The transfected cells were stimulated with FGF2 (100 ng/ml) for 15 min (B) or ephrin-A1 (0.5 μg/ml) for 60 min (C). In separate experiments, the maximal effects of FGF2 and ephrin-A1 were shown to occur at 15 and 60 min, respectively.

Article Snippet: The following antibodies were used according to the manufacturers’ instructions: mouse anti-myc antibody derived from hybridoma MYC1-9E10.2 (ATCC, Manassas, VA, USA; Cat. #CRL-1729), mouse anti-HA antibody derived from hybridoma 12CA5 (Roche, Mannheim, Germany; Cat. #11583816001), mouse anti-Flag M2 monoclonal antibody (clone M2; Sigma-Aldrich Co.; Cat. #F3165), mouse anti-HA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. #sc-7392), rat anti-HA monoclonal antibody (clone 3F10; Roche, Cat. #11867423001), mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology, Cat. #sc-9996); rabbit anti-FGFR1 polyclonal antibody (Santa Cruz, Cat. #sc-121), rabbit anti-FGFR3 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-123), rabbit anti-FRS2α polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8318), rabbit anti-EphA4 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-921), rabbit anti-Akt 1/2 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8312), rabbit anti-phospho-Akt (Ser473) monoclonal antibody (Cell Signaling Technology, Denvers, MA, USA; Cat. #4060), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cell Signaling Technology, Cat. #9101), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cell Signaling Technology, Cat. #9102), mouse anti-phosphotyrosine monoclonal antibody (clone 4G10, Millipore, Billerica, MA, USA Cat. #05–321) and rat anti-nestin antibody (Rat-401, DSHB, Iowa City, IA, USA).

Techniques: Mutagenesis, Binding Assay, Phospho-proteomics, Transfection, Dominant Negative Mutation, Plasmid Preparation

Journal: PLoS ONE

Article Title: Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α

doi: 10.1371/journal.pone.0128826

Figure Lengend Snippet: (A) FGF2-induced activation of FGFR leads to phosphorylation of both Grb2 and Shp2 binding sites on FRS2α. Both the Shp2- and Grb2-mediated pathways were found to lead to activation of MAP kinase via activation of the Ras pathway. Rap1 was not significantly activated. (B) Co-stimulation with FGF2 and ephrin-A1 induced strong and sustained activation of FRS2α. Both the Ras- and Rap1-mediated pathways resulted in quick and robust MAP kinase activation, augmenting self-renewal of NSPCs. (C) Ephrin-A1-induced activation of EphA led to delayed weak activation of MAP kinase, and appeared to be mediated mainly by the Shp2-Ras and Shp2-Rap1 pathways via trans-phosphorylation of FGFR by EphA. Activation of Ras through the EphA-mediated Grb2-Ras pathway was weak. Dotted lines and grey letters indicate decreased signals compared with solid lines and black letters. Two FRS2α molecules are included in the complex to conveniently separate the signal transduction pathways mediated by ephrin/EphA4 and FGF/FGFR. The binding stoichiometry has yet to be studied.

Article Snippet: The following antibodies were used according to the manufacturers’ instructions: mouse anti-myc antibody derived from hybridoma MYC1-9E10.2 (ATCC, Manassas, VA, USA; Cat. #CRL-1729), mouse anti-HA antibody derived from hybridoma 12CA5 (Roche, Mannheim, Germany; Cat. #11583816001), mouse anti-Flag M2 monoclonal antibody (clone M2; Sigma-Aldrich Co.; Cat. #F3165), mouse anti-HA monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Cat. #sc-7392), rat anti-HA monoclonal antibody (clone 3F10; Roche, Cat. #11867423001), mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology, Cat. #sc-9996); rabbit anti-FGFR1 polyclonal antibody (Santa Cruz, Cat. #sc-121), rabbit anti-FGFR3 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-123), rabbit anti-FRS2α polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8318), rabbit anti-EphA4 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-921), rabbit anti-Akt 1/2 polyclonal antibody (Santa Cruz Biotechnology, Cat. #sc-8312), rabbit anti-phospho-Akt (Ser473) monoclonal antibody (Cell Signaling Technology, Denvers, MA, USA; Cat. #4060), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cell Signaling Technology, Cat. #9101), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cell Signaling Technology, Cat. #9102), mouse anti-phosphotyrosine monoclonal antibody (clone 4G10, Millipore, Billerica, MA, USA Cat. #05–321) and rat anti-nestin antibody (Rat-401, DSHB, Iowa City, IA, USA).

Techniques: Activation Assay, Phospho-proteomics, Binding Assay, Transduction