recombinant mouse ephrinb2 fc  (R&D Systems)

Journal: Advanced Science

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

doi: 10.1002/advs.78099

Figure Lengend Snippet: Spinal administration of ephrinB2‐Fc diminishes morphine antinociception. (A, B) Western blot showing that morphine activated ephrinB2‐EphB1 receptor signaling in DRGs (A) and in the spinal cord (B). Left, representative bands. Right, statistical summary. n = 3 in each group. (C, D) Intrathecal administration of ephrinB2‐Fc diminished the analgesic effect of morphine in the tail‐flick test (C) and hot‐plate test (D). Control IgG‐Fc or ephrinB2‐Fc was intrathecally injected 30 min prior to intrathecal injection of morphine. Left, %MPE. Right, AUC. (E–G) Intrathecal administration of ephrinB2‐Fc diminished the analgesic effects of DAMGO (E), but not DPDPE (F), or U69593 (G), in the tail‐flick test (left) and hot‐plate test (right). Control IgG‐Fc and eprhinB2‐Fc were injected intrathecally 30 min before intrathecal administration of DAMGO, DPDPE, or U69593 . Upper, %MPE. Lower, AUC. (C–G) Sample sizes were indicated in brackets. Statistical analysis by unpaired Student's t test in (A) to (G), or two‐way ANOVA with Bonferroni's multiple comparison test in (C) to (G). n.s., no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: DAMGO (HY‐P0210), DPDPE (HY‐P1334), Paroxetine (HY‐122272), and STA‐013 (HY‐179572) were purchased from MedChemExpress; U69593 (U103) was purchased from Sigma; Recombinant mouse ephrinB2‐Fc (496‐EB‐200) was purchased from R&D Systems; control mouse IgG‐Fc (JAC‐015‐000‐008) was purchased from Jackson ImmunoResearch.

Techniques: Western Blot, Tail Flick Test, Hot Plate Test, Control, Injection, Analysis, Comparison

Journal: Advanced Science

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

doi: 10.1002/advs.78099

Figure Lengend Snippet: EphB1 receptors regulate MOR trafficking and control the phosphorylation of MOR at Ser 375 . (A) Schematic illustration of generating Neuro‐2a cells overexpressing MOR. (B) Western blot showing the expression of total protein, plasma membrane (PM) distribution, and cytoplasm distribution of MOR in Neuro‐2a cells overexpressing MOR following treatment of ephrinB2‐Fc (0.01 µg/µL). Left, representative bands. Right, statistical summary. Each experiment was repeated three times. (C) Immunofluorescence staining showing MOR internalization in Neuro‐2a cells overexpressing MOR following treatment of ephrinB2‐Fc (0.01 µg/µL). Left, representative images. The scale bar indicates 10 µm. Right, statistical summary. n = 36 cells in IgG‐Fc + Vehicle group, n = 32 cells in ephrinB2‐Fc + Vehicle group, n = 30 cells in IgG‐Fc + DAMGO group, and n = 47 cells in ephrinB2‐Fc + DAMGO group. (D) Schematic illustration of GRK2‐mediated phosphorylation of MOR at Ser 375 and MOR internalization. MOR agonist induces binding of GRK2, which triggers the phosphorylation of MOR at Ser 375 . β‐arrestin is then recruited and mediates the internalization of MOR. (E) Western blot showing increased phosphorylation of EphB1 receptor and MOR at Ser 375 in Neuro‐2a cells overexpressing MOR following treatment with ephrinB2‐Fc (0.01 µg/µl). Left, representative bands. Right, statistical summary. Each experiment was repeated three times. (F, G) Western blot showing increased phosphorylation of MOR at Ser 375 in DRGs (F) and spinal cord (G) following intrathecal administration of ephrinB2‐Fc (50 ng). Control IgG‐Fc or ephrinB2‐Fc was intrathecally injected 30 min before intrathecal injection of DAMGO or Vehicle. Left, representative bands. Right, statistical summary. n = 3 in each group. (H, I) Western blot showing that knockout of EphB1 receptors in Vglut2 + neurons inhibited DAMGO‐induced phosphorylation of MOR at Ser 375 in DRGs (H) and the spinal cord (I). Left, representative bands. Right, statistical summary. n = 3 in each group. Statistical analysis by unpaired Student's t ‐test in (B), (C), (E), (F), and (G), or one‐way ANOVA with Bonferroni's multiple comparison test in (H) and (I). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: DAMGO (HY‐P0210), DPDPE (HY‐P1334), Paroxetine (HY‐122272), and STA‐013 (HY‐179572) were purchased from MedChemExpress; U69593 (U103) was purchased from Sigma; Recombinant mouse ephrinB2‐Fc (496‐EB‐200) was purchased from R&D Systems; control mouse IgG‐Fc (JAC‐015‐000‐008) was purchased from Jackson ImmunoResearch.

Techniques: Control, Phospho-proteomics, Western Blot, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining, Binding Assay, Injection, Knock-Out, Analysis, Comparison

Journal: Advanced Science

Article Title: EphB1 Receptor Blockade Augments Morphine Antinociception via Inhibiting GRK2‐Mediated µ‐Opioid Receptor Internalization

doi: 10.1002/advs.78099

Figure Lengend Snippet: Activation of EphB1 receptors induces phosphorylation of MOR at Ser 375 and MOR internalization via GRK2. (A) Co‐IP analysis showing increased phosphorylation of GRK2 at tyrosine residues and enhanced interaction between GRK2 and MOR following treatment of ephrinB2‐Fc (0.01 µg/µL) in Neuro‐2a cells overexpressing MOR. The cell lysates were immunoprecipitated with an antibody against GRK2 and immunoblotted with PY99 antibodies to analyze the tyrosine phosphorylation of GRK2, or immunoblotted with Flag antibodies to analyze the interaction between GRK2 and MOR. Left, representative bands. Right, statistical summary. Each experiment is repeated three times. (B) Western blot showing that ephrinB2‐Fc‐induced phosphorylation of MOR at Ser 375 is inhibited by treatment of the GRK2 inhibitor, Paroxetine (5 µM). Upper, representative bands. Lower, statistical summary. Each experiment is repeated three times. (C) Immunofluorescence staining showing that Paroxetine (5 µM) inhibited ephrinB2‐Fc‐induced internalization of MOR in Neuro‐2a cells overexpressing MOR. Left, representative images. Scale bar indicates 10 µm. Right, statistical summary. n = 77 cells in IgG‐Fc + Vehicle group, n = 77 cells in IgG‐Fc + Paroxetine group, n = 65 cells in ephrinB2‐Fc + Vehicle group, and n = 90 in ephrinB2‐Fc + Paroxetine group. Statistical analysis by unpaired Student's t ‐test in (A) or one‐way ANOVA with Bonferroni's multiple comparison test in (B) and (C). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: DAMGO (HY‐P0210), DPDPE (HY‐P1334), Paroxetine (HY‐122272), and STA‐013 (HY‐179572) were purchased from MedChemExpress; U69593 (U103) was purchased from Sigma; Recombinant mouse ephrinB2‐Fc (496‐EB‐200) was purchased from R&D Systems; control mouse IgG‐Fc (JAC‐015‐000‐008) was purchased from Jackson ImmunoResearch.

Techniques: Activation Assay, Phospho-proteomics, Co-Immunoprecipitation Assay, Analysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Comparison

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 promotes tumor immune evasion by upregulating PD-L1 expression within TME (A–C) scRNA-seq analysis of two paired tumor and paratumor samples from LUAD patients showing cell clustering (A), WFDC2 /HE4-expressing cell populations (B), and quantification of WFDC2 /HE4 expression in epithelial/malignant clusters (C). (D) Online dataset analysis of WFDC2 mRNA expression in tumor versus normal LUAD tissues. (E and F) HE4 overexpression (E) or knockout (F) respectively promoted or suppressed the growth of subcutaneous LLC tumors in mice. (G) Survival of mice intraperitoneally inoculated with HE4-overexpressing or control ID8 cells. (H) HE4 knockout attenuated ID8 peritoneal tumor progression, shown by representative abdominal images and ascites volume. (I) SDS-PAGE with Coomassie blue staining showing the purity of Fc and mouse HE4-Fc (mHE4-Fc) recombinant proteins. (J) Administration of mHE4-Fc promoted MC38 subcutaneous tumor growth. (K) mHE4-Fc administration reversed the growth suppression of HE4-KO LLC subcutaneous tumors. (L) mHE4-Fc failed to reverse tumor growth suppression in HE4-KO LLC tumors implanted in Rag1 -deficient mice. (M) Extracellular HE4 did not promote LLC cell proliferation in vitro , as assessed by CCK8 assay. (N) mHE4-Fc administration suppressed CD8 + T cell activation in the TME of HE4-KO LLC tumors, assessed by IFN-γ + and granzyme B + CD8 + T cells. (O) HE4 upregulated PD-L1 expression on macrophages in the microenvironment of LLC, MC38, and HE4-KO LLC tumors. (P–R) mHE4-Fc administration failed to reverse tumor suppression of HE4-KO LLC subcutaneous tumors in Cd274 -deficient mice, with treatment scheme (P), tumor growth (Q), and tumor weight (R). Statistical analyses were performed using Wilcoxon rank-sum test (D), two-way ANOVA (E, F, J–L, and Q), log rank (Mantel-Cox) test (G), and two-tailed paired (C) or unpaired t tests (H, M, N, O, and R). Data represent two independent experiments (E and M).

Article Snippet: Recombinant Fc-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P73252.

Techniques: Expressing, Over Expression, Knock-Out, Control, SDS Page, Staining, Recombinant, In Vitro, CCK-8 Assay, Activation Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 competes with IFN-γ for IFN-γR binding and modulates downstream gene expression (A and B) Raw264.7 cells were stimulated with HE4-Fc (20 μg/mL) or IFN-γ (100 ng/mL) for 3 h, followed by RNA-seq; volcano plots of HE4- (A) or IFN-γ-regulated genes (B) are shown. (C) Genes commonly upregulated by HE4 and IFN-γ. (D) AlphaFold-3-predicted interfaces of HE4-IFNGR1/2 and IFN-γ-IFNGR1/2 complexes, with shared receptor-contact residues highlighted. (E) Competitive binding assay: His-tagged IFNGR1/2 was incubated with Flag-HE4 in the presence or absence of IFN-γ, followed by Ni-TED pull-down and immunoblotting. (F and G) SPR sensorgrams showing binding of mHE4-Fc (F) or mIFN-γ-Fc (G) to mIFNGR1-His. (H and I) ELISA quantification of HE4 and/or IFN-γ levels in ascites from ID8-tumor-bearing mice (H) and LLC-tumor-conditioned media (I). (J) High concentrations of HE4 reduced IFN-γ binding to IFNGR1/2 in competitive pull-down assays. (K) PCA of RNA-seq profiles from Raw264.7 cells treated with HE4-Fc, HE4-Fc plus IFN-γ, or IFN-γ for 12 h. (L) Expression (TPM) of representative STAT1- or STAT3-associated genes following the indicated treatments. Statistical analyses were performed using two-tailed paired Student’s t tests (H and I). Data in (E) and SPR sensorgrams (F and G) are representative of three independent experiments.

Article Snippet: Recombinant Fc-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P73252.

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Competitive Binding Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 blockade promotes antitumor immunity in the tumor microenvironment (A–C) scRNA-seq analysis of LLC tumors from mice treated with control IgG or anti-HE4 antibody ( n = 3 per group), showing UMAP clustering of 26 cell populations (A), relative abundance of each cluster (B), and aggregated cell types (C). ∗ p < 0.05 (D and E) HE4 neutralization reduced Cd274 (PD-L1) expression in myeloid compartments. UMAP feature plots show Cd274 expression in macrophage/monocyte and epithelial/malignant populations (D), with paired comparison across macrophage clusters (E). (F) UMAP visualization of nine intratumoral T cell subclusters in control IgG– and anti-HE4–treated tumors. (G) In the LLC subcutaneous model, intratumoral IFN-γ + and CD69 + CD8 + T cells were quantified by flow cytometry. (H) In the ID8 intraperitoneal model, IFN-γ + CD8 + T cells were quantified by flow cytometry. (I–K) HE4 neutralization failed to suppress LLC tumor growth in Rag1 −/− mice, shown by treatment scheme and tumor growth/endpoint analyses. (L–O) CD8 + T cell depletion abrogated the antitumor efficacy of HE4 blockade, with treatment scheme, tumor growth/endpoint measurements, and confirmation of depletion efficiency by flow cytometry. (P–S) Macrophage depletion using anti-CSF1R diminished the antitumor efficacy of HE4 neutralization, with tumor growth/endpoint measurements and confirmation of depletion efficiency by flow cytometry. Statistical analyses were performed using unpaired t tests (B, C, H, and K), paired t test (E), one-way ANOVA (G, N, O, R, and S), and two-way ANOVA (J, M, and Q). Data in (G, H, and L–S) are pooled from two independent experiments.

Article Snippet: Recombinant Fc-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P73252.

Techniques: Control, Neutralization, Expressing, Comparison, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 neutralization exerts therapeutic activity in human cancer models (A–C) PMA-differentiated THP-1 macrophages were stimulated with Fc or hHE4-Fc, and PD-L1 expression was assessed by flow cytometry (A), immunoblotting (B), and RT-qPCR (C); a commercial hHE4-Fc was included as an independent control. (D) Binding of hHE4 to PMA-differentiated THP-1 cells assessed by flow cytometry. (E and F) PMA-differentiated THP-1 cells were pretreated with ruxolitinib, fludarabine, or Stattic, followed by hHE4-Fc stimulation; PD-L1 was quantified by RT-qPCR (E) and flow cytometry (F). (G and H) Anti-hHE4 monoclonal antibodies inhibited hHE4-induced PD-L1 upregulation in PMA-differentiated THP-1 cells, assessed by RT-qPCR (G) and flow cytometry (H). (I) Anti-hHE4 mAb clone #10 blocked hHE4 binding to PMA-differentiated THP-1 cells. (J) Binding of wild-type or epitope-mutant hHE4-Fc to anti-hHE4 mAb clone #10 was quantified by ELISA. (K) Pharmacokinetic analysis of anti-hHE4 mAb clone #10 in C57BL/6 mice following intravenous administration. (L–O) Fresh human LUAD tumor cell suspensions were treated with anti-hHE4 mAb clone #10, followed by flow cytometric analysis of PD-L1 and ELISA measurement of IFN-γ and granzyme B. (P) Recombinant HE4 suppressed IFN-γ production in human LUAD tumor cell suspensions. (Q–T) HE4 blockade enhanced PBMC-mediated antitumor activity in humanized C-NKG mice bearing OVCAR3 or NCI-H358 tumors, shown by treatment scheme, representative tumors, and tumor weights. Schematics (L and Q) were created using BioRender. Statistical analyses were performed using one-way ANOVA (C, E, and G), paired t tests (M–P), or unpaired t tests (S and T). Data in (A–J) are representative of three independent experiments; data in (Q–T) are pooled from two independent experiments.

Article Snippet: Recombinant Fc-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P73252.

Techniques: Neutralization, Activity Assay, Expressing, Flow Cytometry, Western Blot, Quantitative RT-PCR, Control, Binding Assay, Bioprocessing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Extracellular caspase-1: a critical inducer and a therapeutic target of lung injury in gut ischemia-reperfusion

doi: 10.3389/fimmu.2026.1811868

Figure Lengend Snippet: eCasp-1 binds to TLR4 to drive inflammation, which is effectively suppressed by the novel peptide C16. (A, B) Computational modeling predicted a strong interaction between eCasp-1 (red) and TLR4 (blue). (C) SPR analysis on the binding of eCasp-1 to TLR4 in vitro . (D, E) WT and TLR4 -/- peritoneal macrophages were treated with PBS or eCasp-1 (0.1 µg/ml) for 4 h. (D) IL-6 and (E) TNFα levels in the supernatants were measured by ELISA. (F, G) WT and TLR4 -/- mice received i.p. injections of PBS or eCasp-1 (5 µg/g BW), and plasma was collected 24 h later to measure (F) IL-6 and (G) TNFα. (H) In-silico analysis identified a putative binding site for mouse eCasp-1 (red) on the extracellular domain of TLR4 (blue). (I) C16 (silver), a 16-amino-acid peptide mimic, was designed based on the predicted binding interface and exhibited strong binding to eCasp-1 (red). (J) Computational modeling predicted a potential interaction between the eCasp-1-C16 complex and TLR4. (K) SPR analysis of eCasp-1 binding to TLR4 in the presence or absence of C16. (L) WT peritoneal macrophages were treated with eCasp-1 (0.1 µg/mL) with increasing concentrations of C16 (0.1, 1, 10 µg/mL) for 4 h, and TNFα levels in the supernatants were measured by ELISA. Experiments were repeated 2–3 times and all the data obtained were used for analysis. Data were expressed as mean ± SEM (n = 5–9 samples/group) and compared by one-way analysis of variance and Student-Newman-Keuls method ( * p < 0.05 vs. WT PBS; # p < 0.05 vs. WT with eCasp-1, (+)eCasp-1 (-)C16).

Article Snippet: Recombinant mouse TLR4 (rmTLR4; ≥90% purity) was purchased from R&D Systems (Cat. No. 9149-TR-050, Minneapolis, MN), supplied in carrier-free form, and reconstituted in sterile PBS according to the manufacturer’s instructions.

Techniques: Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, In Silico

Journal: Frontiers in Immunology

Article Title: Extracellular caspase-1: a critical inducer and a therapeutic target of lung injury in gut ischemia-reperfusion

doi: 10.3389/fimmu.2026.1811868

Figure Lengend Snippet: Summary of findings. In gut I/R injury, inflammasomes activation promotes the cleavage of caspase-1 and the extracellular release of its p20 subunit through GSDMD-dependent membrane processes. This release may occur in association with GSDMD pore formation as well as membrane disruption during lytic cell death (pyroptosis). Once released, extracellular caspase-1 (eCasp-1) acts as a potent DAMP by binding to TLR4, thereby amplifying release of inflammatory cytokines, aggravating lung injury. Therapeutic intervention with the inhibitory peptide C16, which specifically blocks the eCasp-1-TLR4 interaction, effectively attenuates systemic inflammation and improves survival outcomes. I/R, Ischemia-reperfusion; GSDMD, Gasdermin-D; eCasp-1, Extracellular caspase-1; DAMP, Damage-associated molecular pattern; TLR4, Toll-like receptor 4.

Article Snippet: Recombinant mouse TLR4 (rmTLR4; ≥90% purity) was purchased from R&D Systems (Cat. No. 9149-TR-050, Minneapolis, MN), supplied in carrier-free form, and reconstituted in sterile PBS according to the manufacturer’s instructions.

Techniques: Activation Assay, Membrane, Disruption, Binding Assay