Journal: eLife

Article Title: An Eya1-Notch axis specifies bipotential epibranchial differentiation in mammalian craniofacial morphogenesis

doi: 10.7554/eLife.30126

Figure Lengend Snippet:

Article Snippet: antibody , Rat monoclonal anti-Jagged1 , DSHB (Iowa, USA) , Ts1.15h, RRID: AB_528317 , 1/300, IHC.

Techniques: Recombinant, Transfection, Protease Inhibitor, In Situ, Sequencing, Cloning, In Situ Hybridization, Construct, Generated, Plasmid Preparation, Mutagenesis, Software

Journal: Experimental and therapeutic medicine

Article Title: Study of dietary‑induced progression of psoriasis‑like mice based on gut macrophage polarization.

doi: 10.3892/etm.2023.11976

Figure Lengend Snippet: Figure 5. Protein expressions of Jagged1/Notch1/HES5 and TLR‑2/IκB‑α/NF‑κB p65 signaling pathway on skin lesions of imiquimod‑induced psoriasis model. (A and a) Western blot analysis of Notch signaling and NRP1 in skin lesions. The mammalian Notch signaling pathway consists of four Notch receptors (Notch 1, 2, 3, and 4) and five Notch ligands (Jagged 1, and 2, and Delta‑like 1, 3 and 4). HES5 is an important downstream target gene of Notch signaling pathway. NRP1 is involved in keratinocyte hyperproliferation of psoriasis accompanied by inflammation activated angiogenesis. (B and b) Western blot analysis of TLR‑2/IκB‑α/NF‑κB p65 signaling in skin lesions. Values are expressed as mean ± SD (*P<0.05, **P<0.01 vs. the normal control; #P<0.05, ##P<0.01, ###P<0.001 vs. the psoriasis group) and were statistically analyzed with one‑way ANOVA. NC, normal control; P, psoriasis; P + SF, psoriasis + SF; SF, stimulating food; p‑, phosphorylated.

Article Snippet: Proteins were transferred onto a PVDF membrane (MilliporeSigma), and then incubated with primary antibodies specific for NF‐κB p65 (cat. no. 6956), phospho‐NF‐κB p65 (cat. no. 3033), Toll‐like receptor‐2 (cat. no. 2229S), IKBα (cat. no. 9242), Jagged‐1 (cat. no. 70109s), Notch‐1 (cat. no. 3608s; all CST; 1:1,000), Hes‐5 (cat. no. ab25374; Abcam; 1:1,000) and anti‐β‐actin (cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.; 1:2,000) overnight at 4 ̊C and then followed by incubation with horseradish peroxidase‐conjugated secondary anti‐mouse or anti‐rabbit IgG (cat. nos.

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Notch Ligand DLL4 Alleviates Allergic Airway Inflammation via Induction of a Homeostatic Regulatory Pathway

doi: 10.1038/srep43535

Figure Lengend Snippet: BALB/c mice were sensitized and challenged with OVA as described. In indicated groups, DLL4 or Jagged1 blocking Ab were given 1 hr before sensitization and challenges. Mice receiving PBS sensitization and challenge and mice given isotype control Ab before OVA sensitization and challenge were used as negative and positive controls, respectively. Serum was collected for Ab detection at one wk after the 2 nd sensitization. Bronchoalveolar lavage and histologic studies of the lungs were performed after aerosol OVA challenge. ( A ) Schematic diagram showing the experimental design of the OVA-induced asthma model. ( B ) NICD expression in nuclear and cytoplasmic extract after stimulation of the splenocytes in the presence of Jagged1 and DLL4 blocking Ab at 10 and 20 μg/mL. ( C ) Serum OVA-specific Ab detected by ELISA; ( D ) Microphotographs showing H&E staining of the lungs; ( E ) Differential counts of BALF leukocytes enumerated by staining characteristics of individual leukocyte populations. A total of 500 cells per cytospin were counted. N = 6–8 mice/group. Data were representative of 4 independent experiments. Comparisons between groups were made by one-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; ( F ) Airway resistance was recorded through an intratracheal cannula while the animals were exposed to increasing concentrations of methacholine (0~60 mg/mL) under anesthesia. N = 6–8 mice/group. Data were representative of 4 independent experiments. Analysis was made by one-way ANOVA. * and # Comparison of OVA group to α-DLL4 and α-Jagged1 groups, respectively; $ α-DLL4 compared to α-Jagged1 group. ** P < 0.01; ***, ### and $$$ P < 0.001.

Article Snippet: When indicated, the stimulation was performed in the presence of anti-DLL4 or anti-Jagged1 Ab (20 μg/mL, BioXCell).

Techniques: Blocking Assay, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: Notch Ligand DLL4 Alleviates Allergic Airway Inflammation via Induction of a Homeostatic Regulatory Pathway

doi: 10.1038/srep43535

Figure Lengend Snippet: CD4 + T cells were isolated from 6 w/o DO11.10 mice and stimulated with OVA 323–339 peptide-pulsed irradiated BALB/c APCs in the presence of Fc-conjugated Jagged1 and DLL4 recombinant proteins. ( A ) Nuclear NICD expression evaluated by Western blotting at 16 hr after stimulation. At 48 hr after stimulation, cells were harvested and analyzed for Th subset development. ( B ) Quantification of main Th subset transcription factors by qPCR; ( C ) cytokine production was detected by intracellular FACS staining. ( Upper ) representative FACS plots showing intracellular cytokine expression of the CD4 + T cells; ( Bottom ) percentage of individual cytokine-expressing CD4 + T-cell subsets. Data represent one of three independent experiments. Comparisons between groups were made by one-way ANOVA. ** P < 0.01; *** P < 0.001.

Article Snippet: When indicated, the stimulation was performed in the presence of anti-DLL4 or anti-Jagged1 Ab (20 μg/mL, BioXCell).

Techniques: Isolation, Irradiation, Recombinant, Expressing, Western Blot, Staining

Journal: Scientific Reports

Article Title: Notch Ligand DLL4 Alleviates Allergic Airway Inflammation via Induction of a Homeostatic Regulatory Pathway

doi: 10.1038/srep43535

Figure Lengend Snippet: CD4 + CD25 + Tregs and CD4 + CD25 - effector T cells (Teff) were isolated from the spleens of DO11.10 mice and stimulated with 200 μg/mL OVA in the presence of α-DLL4 or α-Jagged1 Ab (20 μg/mL). ( A ) Suppression of Teff proliferation by CD4 + CD25 + Tregs in response to OVA stimulation. Data are representative of three separate experiments. Comparison was made by one-way ANOVA. *** P < 0.001. To investigate the induction of pulmonary Tregs, mice were sensitized and challenged with OVA as described in the Methods. When indicated, mice were given control Ig, α-DLL4 or α-Jagged1 Ab via i.v. before OVA sensitization and challenges. ( B ) CD4 + CD25 + FoxP3 + T cells of the lungs after OVA challenge were detected by FACS. Data represented one of three independent experiments. N = 5 mice/group. Comparisons between groups were made by one-way ANOVA. *** P < 0.001. In parallel, mice were given DLL4-overexpressing Raw264.7 (Raw-DLL4, D4) or Raw264.7 transfected with empty vector (Raw-Ctrl, Ctrl) before OVA sensitization and challenges. ( C ) ( left ) DLL4 expression by Raw-DLL4 clones were examined by Western blotting; ( right ) DLL4 expression by the Raw-DLL4 and Raw-Ctrl clones selected for in vivo transfer was quantified by qPCR. To verify the entry of Raw-DLL4 into the lungs, Raw-DLL4 was further overexpressed with GFP as described in the Methods. Pulmonary leukocytes and lung tissues were collected at 30 min and 2hr after transfer. ( D ) ( left ) FACS plots showed GFP-overexpressing Raw-DLL4 before transfer and in the lungs at 2 hr after transfer; ( right ) microphotographs at 2 hr showed the presence of Raw-DLL4 in the lungs as labeled in red by GFP immunostaining. ( E ) Pulmonary CD4 + CD25 + FoxP3 + Tregs after Raw-DLL4 transfer were detected by FACS. Data represented one of two independent experiments. N = 6 mice/group. Comparisons between groups were made by one-way ANOVA. ** P < 0.01; *** P < 0.001.

Article Snippet: When indicated, the stimulation was performed in the presence of anti-DLL4 or anti-Jagged1 Ab (20 μg/mL, BioXCell).

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Clone Assay, Western Blot, In Vivo, Labeling, Immunostaining

Journal: Scientific Reports

Article Title: Notch Ligand DLL4 Alleviates Allergic Airway Inflammation via Induction of a Homeostatic Regulatory Pathway

doi: 10.1038/srep43535

Figure Lengend Snippet: Primer sequences.

Article Snippet: When indicated, the stimulation was performed in the presence of anti-DLL4 or anti-Jagged1 Ab (20 μg/mL, BioXCell).

Techniques: