- 37-8100 - Invitrogen Antibodies EFNB1 antibody

Submitted references EPH receptor B2 stimulates human monocyte adhesion and migration independently of its EphrinB ligands.

Vreeken D, Bruikman CS, Cox SML, Zhang H, Lalai R, Koudijs A, van Zonneveld AJ, Hovingh GK, van Gils JM
Journal of leukocyte biology 2020 Sep;108(3):999-1011

A novel synaptic junction preparation for the identification and characterization of cleft proteins.

Burch A, Tao-Cheng JH, Dosemeci A
PloS one 2017;12(3):e0174895

Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3.

Hruska M, Henderson NT, Xia NL, Le Marchand SJ, Dalva MB
Nature neuroscience 2015 Nov;18(11):1594-605

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Ephrin-B Pan was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Ephrin-B Pan (2D3E9) Mouse Monoclonal Antibody (Product # 37-8100) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry analysis of Ephrin-B was done on SKOV-3 cells (untreated, red histogram) and SKOV-3 cells treated with 40ng/ml TNF alpha for 24 hours (blue histogram). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Ephrin-B Mouse Monoclonal Antibody (37-8100) or with mouse isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10, 000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 PSD-95 is in a complex with ephrin-B3 at synapses a) ST imulated E mission D epletion (STED) imaging of PSD-95 and ephrin-B3 at synapses. DIV21 tdTomato transfected rat cortical neurons were labeled with the indicated antibodies. PSD-95 and ephrin-B3 were imaged at super-resolution using STED (~80 nm). VGlut1 and tdTomato were imaged at conventional resolution (~250 nm) using confocal. Images are high contrast examples of PSD-95, ephrin-B3 and vGlut1 immunostaining. Arrows indicate PSD-95, ephrin-B3 and vGlut1 co-clusters in dendritic spines. Scale bar: 2 um. b) PSD-95, ephrin-B3 and vGlut1 are found in most (>=80%) spines (248 spines on 15 neurons, two separate experiments). c) PSD-95-ephrin-B3 co-clusters are found predominantly in vGlut + spines (162 PSD-95-ephrin-B3 containing spines out of 202 vGlut1 + spines). Single PSD-95 (14 out of 202 vGlut + spines) or ephrin-B3 (20 out of 202 vGlut + spines) clusters are rarely found at synapses. d-f) Co-immunoprecipitation (co-IP) of ephrin-B3 and PSD-95 from P21 wild type and Efnb3 -/- mouse whole brain lysates. The efficacy of ephrin-B3 IP (e) and input (f) are shown for each condition. (g-i) PSD-95-ephrin-B3 co-IP from synaptosomes of wild type and Efnb3 -/- animals (g). Control immunoblots indicating the amount of ephrin-B3 IP (h) and input (i) . (j) PSD-95 selectively co-IPs with ephrin-B3 in synaptosomes isolated from P21 wild type brains despite the efficient IP (k) and presence (l) of each of the three ephrin-B f

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Ephrin-B3 regulates subcellular PSD-95 localization (a) Western blots of non-synaptic (S1), crude synaptosomal (P2) and pure synaptosomal (Syn.) fractions from cortices of P10 and P21 wild type and Efnb3 -/- mice probed with the indicated antibodies. Less synaptic enrichment of PSD-95 is observed in synaptosomes from Efnb3 -/- mice. (b) Model and predicted outcomes for the organotypic slice culture experiment. (c-d) Representative two-photon images of cortical pyramidal neurons from wild type and Efnb3 -/- organotypic slices transfected with PSD-95-GFP and the indicated ephrin-B3 rescue constructs. Right panels in c and d are the high contrast representations of tdTomato and PSD-95-GFP. Filled arrowheads indicate PSD-95-GFP localized to a spine; open arrowheads show PSD-95-GFP localized in the dendritic shaft. PSD-95-GFP is diffusely localized in dendritic shafts of Efnb3 -/- neurons (c) and ephrin-B3 shRNA-transfected wild type neurons (d) . Scale bar in c, d : 5 um. (e) The ratio of average GFP pixel intensities in dendritic shafts to the GFP intensities per pixel in puncta was used to measure the fraction of diffuse PSD-95-GFP in c-d (ANOVA, F (4, 93) = 7.155, p-values: wild type// Efnb3 -/- : 0.0105, wild type// Efnb3 -/- + S332D: 0.0233, Efnb3 -/- // Efnb3 -/- + wt-eB3: 0.0285, Efnb3 -/- // Efnb3 -/- + S332A: 0.0015, Efnb3 -/- + wt-eB3// Efnb3 -/- + S332D: 0.0487, Efnb3 -/- + S332A// Efnb3 -/- + S332D: 0.0027; wild type (n = 22), Efnb3 -/- (n = 31), Efnb3 -/- +

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Ephrin-B3 links MAPK signaling to PSD-95-ephrin-B3 interaction and localization ( a) Sequence alignment of intracellular domains (ICD) of ephrin-B (eB1-3) family. ERK binding D-Domain in the juxtamembrane region of ephrin-B3 (yellow box) and a putative MAPK phosphorylation motif (green box). Red boxes indicate the positions of point mutations in the D-domain (L293A) and in the putative MAPK phosphorylation motif (S332A/S332D). ( b) Immunoprecipitation, using antibodies to Flag, of HEK 293T lysates transfected with PSD-95-GFP and indicated flag-tagged ephrin-B3 (flag-eB3) constructs. (c) Quantification of co-IP experiments shown in b (ANOVA, F (3, 23) = 9.842. WT-S332D: p = 0.0475, WT-L293A: p = 0.0034, S332A-S332D: p = 0.0087, S332A-L293A: p = 0.0006; n = 6 blots from separate transfections). (d) In vitro generated flag-tagged wild type and mutant ephrin-B3 ICDs used to pull down PSD-95-GST. Direct interaction between the two proteins is disrupted by S332D ephrin-B3 ICD. (e) Quantification of pull-down experiments from d (ANOVA, F (2, 9) = 2.823, Fisher's LSD post-hoc, p = 0.0176 (WT-S332D), p = 0.0257 (S332A-S332D); n = 4 independent in vitro binding experiments). (f) Representative high contrast STED images of DIV21 neurons transfected with tdTomato, ephrin-B3 (eB3) shRNA and indicated flag-eB3 rescue constructs. Arrowheads indicate co-localized, flag-ephrin-B3/PSD-95/vGlut1 clusters. Scale bars : , 1 um. (g) Quantification of spine localization of flag-ephrin

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 ERK phosphorylation of S332 regulates subcellular localization of ephrin-B3 (a) Schematic representation of the experiments. (b) Representative immunoblots of lysates from DIV14 rat cortical neurons treated with TTX (1uM, 4 hr.) and U0126 (10uM, 4 hr.) to block activity and ERK activation, respectively. Depolarization with KCl (55mM, 1 hr.) was used to elevate ERK activity. ( c ) Ratio of pS332 to ephrin-B3 signals was used to quantify the effect of neuronal activity on S332 phosphorylation (ANOVA, F (3, 12) = 19.27, Tukey's post-hoc, TTX-KCl: p = 0.0012, TTX+U0126-KCl: p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 2 FIGURE Increased expression of EphrinB in progressive human atherosclerotic lesions . ( A ) Expression heatmap of Ephrin family ligands in endothelial cells and vascular smooth muscle cells. Blue indicates lower and red higher expression. ( B ) EphrinB1/B2/B3 expression in HUVECs stimulated with 20 ng/mL IL-1beta or 10 ng/mL TNF-alpha for 24 h. Results are relative to untreated cells, set as 1. Mean +- sem of n = 3. * P < 0.05. ( C ) Overview pictures of immunohistochemical staining for EphrinB in human aortic sections in different stages of atherosclerosis. Lower images show higher power magnification of the arrow-indicated fields. Scale bars represent 350 and 50 um, respectively. ( D-F ) Quantification of EphrinB signal on total area ( D ), adventitia, media, and intima regions ( E ) and total plaque area ( F ). Results are relative to stage I, set as 1. Mean +- sem of n = 6. ( D-F ) * P < 0.05 compared to indicated stages. ( E ) * P < 0.05 representing interstage variability within a region compared to stage I and # P < 0.05 representing intrastage variability between regions. Stage I: normal, adaptive intimal thickening, intima xantoma; II: pathologic intimal thickening, early fibroatheroma; III: late fibroatheroma, thin cap fibroatheroma, ruptured plaque; IV: healing rupture, fibrous calcifies plaque

37-8100

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