recombinant human ephb4 fc chimera protein  (R&D Systems)

Journal: Journal of Nanobiotechnology

Article Title: EphB4-decorated biomimetic nanoparticles enhance osteoclast targeting and therapeutic effect in osteoporosis

doi: 10.1186/s12951-026-04379-1

Figure Lengend Snippet: Characterization and evaluation of E4-Bil PNPs targeting OCs through EphB4. ( A ) Fluorescence images of GFP-tagged EphB4 (GFP-E4) in Control OBs (Ctrl-OB) and GFP-E4 overexpressing OBs (E4-OB). ( B ) Histogram of GFP-E4 fluorescence intensity in Ctrl-OB and E4-OB, analyzed by flow cytometry. The bar graph indicates the percentage of GFP-positive cells. Data represent the mean ± s.e.m. ( n = 3). ( C ) Gene expression confirming upregulated EphB4 expression. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using an unpaired t-test (**** p < 0.0001). ( D ) Protein expression of EphB4, Caveolin-1, and β-actin in the cytoplasm and cell membrane, assessed by Western blotting. ( E ) Quantitative analysis of EphB4 protein levels. ( F ) Encapsulation efficiency of bilirubin in Bil-PNPs, indicating the proportion of bilirubin successfully encapsulated. Data represent the mean ± s.e.m. ( n = 3). ( G ) Particle size of PNPs, Bil-PNPs, CMNVs, and E4-Bil-PNPs measured by DLS. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (** p < 0.01, **** p < 0.0001). ( H ) Zeta potential of PNPs, Bil-PNPs, CMNVs, and E4-Bil-PNPs measured by DLS. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (* p < 0.05, *** p < 0.001, **** p < 0.0001). ( I ) EF-TEM images showing immunogold staining of EphB4 (yellow arrows) visualized with a secondary antibody. Scale bar = 100 nm. ( J ) Bilirubin release profiles over 8 days for Bil-PNPs and E4-Bil-PNPs, demonstrating sustained release. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using two-way ANOVA (**** p < 0.0001)

Article Snippet: For transfection, 2.5 μg plasmid DNA encoding EphB4 (Origene, USA) was diluted in 125 μL α-MEM.

Techniques: Fluorescence, Control, Flow Cytometry, Gene Expression, Expressing, Membrane, Western Blot, Encapsulation, Zeta Potential Analyzer, Staining

Journal: Journal of Nanobiotechnology

Article Title: EphB4-decorated biomimetic nanoparticles enhance osteoclast targeting and therapeutic effect in osteoporosis

doi: 10.1186/s12951-026-04379-1

Figure Lengend Snippet: Specificity of E4-Bil PNPs for OC targeting. ( A ) Fluorescence images from the uptake study showing internalization of RhB-PNPs (Rhodamine B encapsulated in PLGA) and E4-RhB-PNPs (RhB encapsulated in PLGA and coated with EphB4-overexpressing cell membranes) into OCs. Scale bar = 100 μm. ( B ) Quantification of E4-RhB-PNP uptake in OCs, confirming efficient and targeted internalization. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (**** p < 0.0001). ( C ) Fluorescence images of EFNB2 expression in OCs during differentiation on days 0, 3, and 5. Scale bar = 50 μm. ( D ) Quantification of EFNB2 expression levels on days 0, 3, and 5, showing temporal changes during osteoclastogenesis. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (*** p < 0.001). ( E ) Fluorescence images of E4-Bil-PNP uptake on days 0, 3, and 5, demonstrating their interaction with OCs throughout differentiation. Scale bar = 100 μm. ( F ) Quantification of E4-Bil-PNP uptake across the differentiation timeline (days 0, 3, and 5). Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (a, compared with day 0; b, compared with day 3; *** p < 0.001, **** p < 0.0001). ( G ) Fluorescence imaging of the EFNB2 inhibition assay showing reduced uptake of E4-RhB-PNPs following pre-treatment with EFNB2 antibody for 1 h, validating EFNB2-dependent targeting. Scale bar = 50 μm. ( H ) Quantification of E4-RhB-PNP uptake under EFNB2 inhibition conditions, confirming the specificity of the interaction. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using an unpaired t-test (** p < 0.001). ( I ) Western blot analysis of EFNB2 expression in various cell types, including OCs, MSCs, macrophages (MΦ), and OBs. ( J ) Fluorescence images of E4-RhB-PNP uptake in these cell types. Scale bar = 100 μm. ( K ) Quantification of E4-RhB-PNP uptake in the different cell types. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (**** P p < 0.0001). ( L ) Schematic illustration of the transendothelial penetration assay to evaluate the ability of E4-RhB-PNPs to traverse cellular barriers. ( M ) Fluorescence images of RhB-PNP and E4-RhB-PNP uptake under inflammatory conditions. Scale bar = 50 μm. ( N ) Quantification of uptake under inflammatory conditions, highlighting the role of inflammation in enhancing NP penetration. Data represent the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA (*** p < 0.001 and **** p < 0.0001). ( O ) Fluorescent imaging of the uptake comparison of RhB-PNPs and E4-RhB PNPs under non-inflammatory conditions. Scale bar: 100 μm. ( P ) Quantification of the uptake under non-inflammatory conditions. Data represents the mean ± s.e.m. ( n = 3) and were analyzed using one-way ANOVA

Article Snippet: For transfection, 2.5 μg plasmid DNA encoding EphB4 (Origene, USA) was diluted in 125 μL α-MEM.

Techniques: Fluorescence, Expressing, Imaging, Inhibition, Western Blot, Comparison

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative image of ACKR1-positive lung vessel from LMB-bearing mouse 28 days after implantation, showing colocalization of ACKR1 with venous endothelial marker EphB4. CD31 = magenta, EphB4 = yellow, ACKR1 = green. All ACKR1 pos vessels also expressed EphB4. 22.7±1.4% of EphB4 pos venules expressed ACKR1. Quantitation shown in . (B) Representative annotated IF image for QuPath-based proximity analysis of tumor cell distance to nearest venule (EphB4 = yellow, Pan-CK = tumor cell marker pancytokeratin in red, ACKR1 = green, and DAPI = blue). For QuPath annotations, yellow line = vessel border, red circle = disseminated tumor cell (DTC), red line = shortest distance between DTC and nearest venule border. (C) Quantification of the number of DTCs within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 116 ACKR1 pos venules, n = 100 ACKR1 neg venules, across 4 individual mice). Unpaired t-tests. Summed data shown here for clarity, individual data points shown in . (D) Percentage of ACKR1-positive and ACKR1-negative venules with at least one DTC within 50 µm of the venule border. Chi-squared test. (E) Representative IF images for QuPath proximity analysis of neutrophil distance to nearest venule, as described in panel B except Ly6G = neutrophil marker (cyan) and red lines indicate shortest distance between neutrophil and nearest venule border. (F) Quantification of the number of neutrophils within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 16 ACKR1 pos venules, n = 16 ACKR1 neg venules, across 3 individual mice). Unpaired t-tests. Summed data shown here for clarity, individual data points shown in .

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Marker, Quantitation Assay

Journal: bioRxiv

Article Title: Endothelial ACKR1 expression regulates neutrophil infiltration and breast cancer metastatic engraftment in the lung metastatic niche

doi: 10.64898/2026.03.15.711832

Figure Lengend Snippet: (A) Representative low-magnification images of lung from mice implanted with LMB tumors at Day 28, showing overall distribution of ACKR1 expression relative to EphB4 expression. (B) Quantification of vessels positive for ACKR1, EphB4, and both ACKR1/EphB4, across 4 individual mice. (C) Quantification of the number of DTCs within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 116 ACKR1 pos venules, n = 100 ACKR1 neg venules, across 4 individual mice). Unpaired t-tests. Individual data points from graphs in . (D) Quantification of the number of neutrophils within indicated distance from the border of ACKR1-positive or ACKR1-negative venules in Day 28 LMB-bearing mouse lungs (n = 16 ACKR1 pos venules, n = 16 ACKR1 neg venules, across 3 individual mice). Unpaired t-tests. Individual data points from graphs shown in .

Article Snippet: Primary antibodies used included anti-mouse ACKR1 (1 μg/mL, a kind gift of Dr. Ulrich von Andrian, Harvard Medical School), anti-mouse CD31 (1:100, Cell Signaling, 77699), anti-mouse EphB4 (2.5 μg/mL, R&D Systems, AF446), anti-mouse Pan Cytokeratin (1:200, ThermoFisher/Bioss, BS-10403R), and anti-mouse Ly-6G (E6Z1T) (1:200, Cell Signaling, 87048).

Techniques: Expressing