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: 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

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–L , Cresyl violet stain of coronal brain sections from control (A–D), Nestin ; Epha4 fx/fx (E–H), and GFAP ; Epha4 fx/fx (I–L) mice. Shown are the corresponding sections of these mice along the antero-posterior axis of the telencephalon. The Nestin ; Epha4 fx/fx cortex appears smaller than the control cortex, while the GFAP ; Epha4 fx/fx cortex is almost the same size as the control cortex. M–P, Quantification of cortical thickness (M, O) and proliferating zone (VZ+SVZ) width (N, P) in Nestin;Epha4 fx/fx (M, N) and GFAP;Epha4 fx/fx (O, P) mice. Solid lines in control animals (A to C) and corresponding lines in mutants (E–G, I–K) indicate the antero-posterior location where these thicknesses were measured. Enlargements of the measured areas in the anterior part (A, E, and I) are shown in the rightmost column (D, H, and L, respectively). Cortical thickness was reduced in all brain sections from Nestin ; Epha4 fx/fx mice, and the proliferating zone width was thinner only in anterior sections. However, in GFAP ; Epha4 fx/fx mice, cortical thickness and proliferating zone width were similar to controls. A, anterior; M, middle; P, posterior. N = 5 for each measurement, (*) P < 0.05. Error bars represent SD. Scale bar, 500 μm.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–F , Immunohistochemical EphA4 expression pattern in control (A–C) and Nestin ; Epha4 fx/fx mouse brain (D–F). Expression of EphA4 in Nestin ; Epha4 fx/fx mouse forebrain starts to disappear after E11.5. G–L, Immunohistochemical EphA4 expression pattern in control (G–I) and GFAP ; Epha4 fx/fx mouse brain (J–L). Expression of EphA4 in GFAP ; Epha4 fx/fx mouse cortex starts to diminish after E13.5 but does not disappear completely in this mutant. M, N, western blot analysis of EphA4 expression in the cortex of control and Nestin;Epha4 fx/fx mice (M) or GFAP;Epha4 fx/fx mice (N). Western blotting results are consistent with those of immunohistochemistry. c, control; m, Nestin;Epha4 fx/fx in panel M and GFAP;Epha4 fx/fx in panel N. Scale bars, 100 μm.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Immunohistochemical staining, Expressing, Control, Mutagenesis, Western Blot, Immunohistochemistry

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–I , Anterior coronal sections from mutant and control mice were immunostained for a marker of RGCs (PAX6, green) and for a marker of young differentiated neurons (βIII tubulin [TUBB3], red). Representative sections at E14.5, E17.5, and P0 are shown on the left. J–Q , Quantified thickness of the RGC layer (PAX6+, green) (J, K), the number of RGCs (L, M), the differentiated neuronal area (TUBB3+, red) (N, O), and the number of neuronal cells (P, Q) using these stained sections ( Nestin;Epha4 fx/fx , J, L, N, P; GFAP;Epha4 fx/fx , K, M, O, Q). The PAX6-expressing progenitor cell population in the VZ was of comparable size in both mutants and controls from E13.5 to E15.5 (A–C, J–M), but was considerably smaller at E17.5 and P0 in Nestin;Epha4 fx/fx mice (D, E, G, H, J, L). Both mutants showed normal cortex at E13.5, but the cortical layer size of post-mitotic neurons (TUBB3+) became thicker at E14.5 compared to controls (A–C, N–Q). The neuronal area in mutants returned to the size of controls at E15.5 (D–F, N–Q). However, in Nestin;Epha4 fx/fx mice, the neuronal area was considerably smaller at P0, while in GFAP;Epha4 fx/fx mice it was not (G–I, N–Q). N = 5, (*) P < 0.05. Error bars represent SD. Scale bar, 100 μm.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Mutagenesis, Control, Marker, Staining, Expressing

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–C, Representative image of apoptotic cells in the cortex of control (A), Nestin;Epha4 fx/fx (B), and GFAP;Epha4 fx/fx (C) mice. Apoptotic cells were detected by TUNEL staining at E14.5. Apoptotic cells (black arrowheads) show intense dark black staining, whereas blood cells (red arrow head) exhibit light brown staining in the cytoplasm. The boxed areas are magnified (lower right insets) to show apoptotic cells. The number of apoptotic cells between the dotted lines in each section was counted. D and E , Quantification of the number of apoptotic cells detected in panels A, B, and C. The percentage of apoptotic cells per all cells between the dotted lines in Nestin;Epha4 fx/fx (D) and GFAP;Epha4 fx/fx (E) mice is shown. There was no significant difference in the percentage of apoptotic cells. N = 4 per genotype. Error bars represent SD. Scale bar, 100 μm.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Control, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–I, Anterior sections isolated from mutants and control mice between E13.5 and P0 were immunostained for an INP marker (TBR2, green). J and K, The number of TBR2+ cells within 100-μm-wide regions of Nestin;Epha4 fx/fx (J) and GFAP;Epha4 fx/fx (K) mouse cortex. At E14.5, TBR2 immunostaining revealed overproduction of INPs in both mutants compared to controls (A–C, J, K), suggesting that there may be accelerated cortical neurogenesis around E14.5 in both mutants. However, at E17.5 and P0, TBR2+ cells were depleted in Nestin;Epha4 fx/fx mice (E, H, E17.5 and P0 in J) but the numbers were unaffected in GFAP;Epha4 fx/fx mice compared to controls (F, I, E17.5 and P0 in K). L–W, Co-staining for pHH3 and PAX6 or TBR2. Mitotic cells were immunostained for pHH3 (red). The pHH3+ cells were co-stained with PAX6 (green), a marker for RGCs, or with TBR2 (green), a marker of INPs. Co-stained cells are shown by yellow and white arrowheads, respectively. X and Y, Quantification of the number of pHH3+ cells co-labeled by either marker in Nestin;Epha4 fx/fx (X) and GFAP;Epha4 fx/fx (Y) mice. The number of pHH3+PAX6+ RGCs (P6) and pHH3+TBR2+ INPs (T2) was counted separately at E13.5, E14.5, E15.5, and E17.5 in control, Nestin;Epha4 fx/fx , and GFAP;Epha4 fx/fx mice. More INPs (pHH3+TBR2+) are dividing at E14.5 in both mutants. Nestin;Epha4 fx/fx exhibited fewer mitotic RGCs (pHH3+PAX6+) than controls at E17.5, whereas GFAP;Epha4 fx/fx mice showed no difference from controls. P6, pHH3+PAX6+; T2, pHH3+TBR2+. N = 5 (A–K) and 4 (L–Y), (*)P < 0.05. Error bars represent SD. Scale bars: A–F, 50 μm, G–I and L–W, 100 μm.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Isolation, Control, Marker, Immunostaining, Staining, Labeling

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–J, Evaluation of the population of cells that re-enter and exit the cell cycle in Nestin;Epha4 fx/fx and GFAP;Epha4 fx/fx mice between E13.5 and E15.5. BrdU was administered to pregnant mothers 24 h before collecting E14.5 and E15.5 embryos. Coronal sections of the control and mutant cortices were co-stained for BrdU and Ki67 to detect the cell population that re-enters the cell cycle and co-stained for BrdU and TUBB3 to mark the cell population that exits the cell cycle. Ki67+BrdU+ cells represent proliferating cells that include RGCs and INPs, while TUBB3+BrdU+ cells represent differentiated neuronal cells. Representative immunofluorescent images at E15.5 for BrdU (green) and Ki67 (red) (A–C), and those for BrdU (green) and TUBB3 (red) (D–F). Quantification of the number of Ki67+BrdU+ cells (G, H) and TUBB3+BrdU+ cells (I, J) in Nestin;Epha4 fx/fx (G, I) and GFAP;Epha4 fx/fx (H, J) mice. In both mutants, the Ki67+BrdU+ cell to total BrdU+ cell ratio decreased around E14.5, whereas the TUBB3+BrdU+ cell to total BrdU+ cell ratio significantly increased relative to controls. These findings suggest that differentiation of RGCs and INPs to neurons is accelerated at this stage in mutants relative to controls. These changes are expressed quantitatively as the fraction of double stained cells relative to the total number of BrdU+ cells in the cortex. N = 5 per genotype, (*) P < 0.05. Error bars represent SD. Scale bars, 50 μm. K and L, Neurosphere formation from cortical cells isolated from Nestin;Epha4 fx/fx cortex in the presence of FGF2 alone, clustered ephrin-A1 alone, or FGF2 plus clustered ephrin-A1. Representative images of neurosphere formation (K) and the number of neurospheres in K (L). Fewer neurospheres grew from isolated Nestin;Epha4 fx/fx cortical cells than from control cortical cells. FGF2 increased the number of the neurospheres derived from control E14.5 cortex but not that from mutant E14.5 cortex, whereas clustered ephrin-A1 alone, a ligand for EphA4, did not affect neurosphere growth. M–P, Differentiation of neurospheres derived from control and Nestin;Epha4 fx/fx cortical cells. The cells from the E14.5 cortex were cultured for 4–5 days to form primary neurospheres. Cells from the primary neurospheres were replated to obtain a monolayer culture and used for immunostaining as described in the Materials and Methods. Representative image of neurospheres immunostained with markers for INPs (TBR2, red) and neurons (TUBB3, green) (M). There was a higher percentage of spheres with TBR2+ (white arrowheads) and TUBB3+ (yellow arrowheads) cells in the mutant (lower figure in M) than in controls (upper figure in M). Representative western blot analysis of TBR2 and TUBB3 in neurospheres from control and Nestin;Epha4 fx/fx mice (N) and quantification of TBR2 (O) and TUBB3 (P) signal intensities in neurospheres from control and Nestin;Epha4 fx/fx mice. The level of TBR2 and TUBB3 expression in the spheres increased in mutants compared to controls, showing that spheres of Nestin;Epha4 fx/fx are in a more differentiated state compared to control. N = 5 per genotype, (*) P < 0.05. Error bars represent SD.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Control, Mutagenesis, Staining, Isolation, Derivative Assay, Cell Culture, Immunostaining, Western Blot, Expressing

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A–L, Representative immunofluorescent images showing the cortical distribution of BrdU-labeled cells (green) and markers (red) for layer 5 (CTIP2) and layers 2/3 (CUX1). Layer names are shown on the left of the images. M–P, Quantified distribution of BrdU+ cell numbers in 10 bins spanning the layers of the cortex. BrdU was injected to label proliferative cells that differentiate into migrating neurons at E13.5 (A–C, G–I, M, O) and E15.5 (D–F, J–L, N, P). The cortical distributions of these cells were analyzed at P0 in control (A, D, G, J), Nestin;Epha4 fx/fx (B, E, H, K) and GFAP;Epha4 fx/fx (C, F, I, L) mice. In Nestin;Epha4 fx/fx mice, a greater fraction of neurons born at E13.5 (M, red bar) was found in upper cortical layers compared to controls (M, blue bar), but a smaller fraction of cells born at E15.5 (N, red bar) was found in upper layers compared to controls (N, blue bar). In contrast, GFAP;Epha4 fx/fx mice exhibited a greater fraction of neurons born at both E13.5 and E15.5 (O and P, yellow bar) in upper cortical layers compared to controls (O and P, green bar). N = 3 per genotype, (*) P < 0.01. Error bars represent SD.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Labeling, Injection, Control

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A and B, Total number of cells labeled with the layer marker CTIP2 or CUX1 at P0 in Nestin;Epha4 fx/fx (A) or GFAP;Epha4 fx/fx (B) mice compared to controls. C – F , The ratio of the number of CTIP2+BrdU+ or CUX1+BrdU+ cells relative to the total number of BrdU+ cells. BrdU was injected to label proliferative cells at E11.5, E13.5, E15.5, and E17.5 in Nestin;Epha4 fx/fx (C, E), GFAP;Epha4 fx/fx (D, F) and corresponding control mice. Both the mutant and the control embryos show a similar cortical layer structure. However, the total number of CUX1+ cells at P0 decreased in Nestin;Epha4 fx/fx (A) but increased in GFAP;Epha4 fx/fx (B) mice compared to controls. In Nestin;Epha4 fx/fx mice, a larger number of CTIP2+ cells was co-labeled with BrdU at E11.5 but fewer were co-labeled at E13.5 compared to controls (C), whereas in GFAP;Epha4 fx/fx mice, the number of co-labeled cells was similar to that of controls at both E11.5 and E13.5 (D). The number of CUX1+ cells co-labeled with BrdU at E13.5 was greater in both mutants (E, F), while the number at E15.5 was lower in Nestin;Epha4 fx/fx mice (E) and greater in GFAP;Epha4 fx/fx mice compared to controls (F). IZ, intermediate zone; SP, subplate. N = 6 for total number of CUX1+ cells and N = 3 for other markers per genotype, (*) P < 0.01. Error bars represent SD.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Labeling, Marker, Injection, Control, Mutagenesis

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: A and B, Representative western blot analysis of ERK1/2 phosphorylation (pERK1/2) in cortical cells from control and Nestin;Epha4 fx/fx mice (A) or GFAP;Epha4 fx/fx mice (B). Dissociated cells from the E15.5 telencephalon were stimulated for 20 min with FGF2 after 1 h starvation and lysed immediately. Stimulated and non-stimulated cells are marked by + or—, respectively. Total ERK1/2 expression (ERK) was used to show equal sample loading. C and D, Quantification of pERK1/2 and ERK1/2 signal intensities in the cortical cells stimulated with FGF2 (+) or not (-) in Nestin;Epha4 fx/fx (C) and GFAP;Epha4 fx/fx (D) mice. E and F, Representative western blot analysis of FRS2α phosphorylation (pFRS2α) in cortical cells from control and Nestin;Epha4 fx/fx mice (E) or GFAP;Epha4 fx/fx mice (F). Cells from the E15.5 telencephalon were examined as described above. GAPDH expression was used to show equal sample loading. G and H, Quantification of pFRS2α and GAPDH signal intensities in cortical cells stimulated with FGF2 (+) or not (-) in Nestin;Epha4 fx/fx (G) and GFAP;Epha4 fx/fx (H) mice. ERK1/2 and FRS2α phosphorylation levels were much lower in both mutants compared to controls. Molecular size markers are shown in kDa on the right side of the western blot lanes. N = 5 per genotype, (*) P < 0.05. Error bars represent SD.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: Western Blot, Phospho-proteomics, Control, Expressing

Journal: PLoS ONE

Article Title: EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

doi: 10.1371/journal.pone.0126942

Figure Lengend Snippet: In early brain corticogenesis, EphA4 in collaboration with FGFRs regulates transition through the neuronal lineage and accelerates self-renewal of RGCs. Black and gray arrows indicate the process of differentiation and self-renewal, respectively. Red arrow-headed lines indicate the function of acceleration and blue T-shaped lines indicate that of suppression. PAX6, TBR2, and TUBB3 are the markers for RGCs, INPs, and neuronal cells, respectively. Gray circles, RGCs; Green circle, committed RGCs; Green squares, INPs; Blue triangles, neuronal cells. CP, cortical plate; SVZ, subventricular zone; VZ, ventricular zone.

Article Snippet: Antibodies used for western blotting were as follows: rabbit anti-EphA4 polyclonal antibody (Cat. #sc-921; 1:2000, Santa Cruz), rabbit anti-FRS2α polyclonal antibody (Cat. #sc-8318; 1:1000, Santa Cruz), rabbit anti-GAPDH (Cat. #sc-25778, 1:3000, Santa Cruz), rabbit anti-phospho-p44/42 polyclonal mitogen-activated protein kinase (MAPK) (ERK) (Thr202/Tyr204) antibody (Cat. #9101; 1:4000, Cell Signaling Tech.), rabbit anti-p44/42 MAPK (ERK) polyclonal antibody (Cat. #9102; 1:4000, Cell Signaling Tech.), and rabbit anti-pFRS2α(Y196) (Cat. #3861; 1:1000, Cell Signaling Tech.).

Techniques: