mouse anti necd antibody  (Developmental Studies Hybridoma Bank)

Journal: The Journal of Cell Biology

Article Title: Drosophila Abi maintains blood cell homeostasis by promoting clathrin-mediated endocytosis of Notch

doi: 10.1083/jcb.202505091

Figure Lengend Snippet: Abi-dependent CME is required for ligand-induced Notch internalization. (A and B) Single confocal slices of primary hemocytes from HmlΔ-GAL4 /+ and UAS-abi RNAi /+; HmlΔ - GAL4 /+ ( HmlΔ > abi RNAi ) late-third instar larvae. Hemocytes were pulsed with Alexa Fluor 555-mBSA (mBSA, a CME marker) and 70 kDa FITC-dextran (Dex70, a macropinocytosis marker) (A) or 10 kDa FITC-dextran (Dex10, a GEEC endocytosis marker) alone (B) for 5 min, chased for 5 min, and stained with DAPI. (C) Quantification of endocytic events in A and B. The ratios of mBSA, Dex70, and Dex10 to DAPI fluorescence intensities are presented as percentages of HmlΔ-GAL4 /+. n = 30 hemocytes. (D) Single confocal slices of S2N cells transfected with abi , Chc , or Rabankyrin dsRNA. Transfected S2N cells were pretreated with 0.7 mM CuSO 4 and cocultured with S2S cells. Live cocultured cells were incubated with an anti-NECD antibody at 4°C for 30 min to prelabel surface Notch receptors (on S2N cells) and further incubated at 25°C for 30 min to allow endocytosis to resume. After fixation, cocultured cells were sequentially stained for surface (green) and internalized (pseudocolored magenta) Notch receptors under nonpermeant and permeant conditions, respectively. Permeabilized cocultured cells were additionally stained for Myc-Ser (blue). Arrowheads indicate intracellular punctate structures containing internalized Notch. (E) Quantification of the ratio of internal to surface Notch fluorescence intensities. n = 9 cells. (F) Single confocal slices of S2N cells transfected with shi dsRNA, pretreated with 0.7 mM CuSO 4 for 24 h, and cocultured with S2S cells for 6 h. Notch receptors on S2N cells were prelabeled with an anti-NECD antibody and allowed to internalize as in D. After fixation and permeabilization, cells were stained for Flag-Chc (green), HA-Abi (pseudocolored magenta), and prelabeled Notch (blue). Arrowheads indicate colocalization of Flag-Chc, HA-Abi, and Notch. Data represent the mean ± SEM. Statistical analyses were performed using Student’s t test (C) or by a one-way ANOVA with the Tukey–Kramer post hoc test (E). Comparisons are with HmlΔ-GAL4 /+ in C and mock-transfected isolated (-Ser) S2N cells in E (***P < 0.001). Scale bars: 5 μm (A and B); 10 μm (D and F).

Article Snippet: Live cocultured cells were incubated in serum-free M3 medium at 4°C with 5 μg/ml mouse anti-NECD antibody (C458.2H; DSHB) for 30 min to label surface Notch receptors.

Techniques: Marker, Staining, Fluorescence, Transfection, Incubation, Isolation

Journal: Science Advances

Article Title: 12R-HETE acts as an endogenous ligand for Nur77 in the intestines and regulates NKp46 + ILC3 development

doi: 10.1126/sciadv.adz8405

Figure Lengend Snippet: ( A ) Percentages of Ki67 + cells among RORγt + ILC3s are shown on the left. Absolute numbers of Ki67 + cells among RORγt + ILC3s are shown on the right. ( B ) Percentages of Ki67 + cells among NKp46 + ILC3s in the intestinal lamina propria. ( C ) Percentages of Ki67 + cells among NKp46 − ILC3s. ( D ) Numbers in flow plots represent percentages of T-bet + NKp46 + cells in the ILC3 gate. ( E ) Percentage of T-bet + cells among NKp46 + ILC3s. ( F ) IFN-γ MFI among NKp46 + ILC3s. ( G ) Percentage of T-bet + cells among DN ILC3s. ( H ) IFN-γ MFI among NKp46 + ILC3s. ( I ) Experimental design for the treatment of 12R-HETE and DAPT. SI LPLs were isolated from the 2-week-old mice. ( J ) Percentages and numbers of NKp46 + ILC3s were analyzed by flow cytometry after Notch signaling inhibitor treatment. ( K ) NKp46 MFI among NKp46 + ILC3. Data are means ± SEM; n = 5 to 6. Each point represents a biological replicate. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; n.s., not significant.

Article Snippet: In mechanistic experiments, mice were intraperitoneally injected with Notch inhibitors DAPT [MedChemExpress (MCE)] or Impdh1 inhibitors MPA (MCE) after treatment with PBS or 12R-HETE, and animals were euthanized after 10 days.

Techniques: Isolation, Flow Cytometry

Journal: Journal of Advanced Research

Article Title: An injectable nano-hydroxyapatite-incorporated hydrogel with sustained release of Notoginsenoside R1 enhances bone regeneration by promoting angiogenesis through Notch1/Akt signaling

doi: 10.1016/j.jare.2025.05.025

Figure Lengend Snippet: (A) The angiogenic-related gene expression quantified by qRT-PCR at day 4, day 7 and day 10. (B) The angiogenic proteins of VEGF and VEGFR-2 analyzed by western blot. (C)semiquantitative analysis of VEGF and VEGFR-2 protein expression. (D) The critical markers Notch1, Hes1 involved in the Notch 1 pathway analyzed by western blot and their semiquantitative analysis (E).(F)Western blot and semiquantitative analysis (G) of Akt protein expression.(H)The gene expression of Akt and VEGF under NGR1 stimulation with Notch 1 inhibition. (I) The gene expression of Notch 1 and VEGF under NGR1 stimulation with Akt inhibition. (J) Western blot and semiquantitative analysis (K) of Akt protein expression induced by NGR1 under Notch 1 inhibition. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Article Snippet: Subsequently, Notch 1 inhibitor (HY-1302, MCE, USA) and Akt inhibitor (HY-10358, MCE, USA) were applied to evaluate their effects on angiogenic-related gene expression.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Expressing, Inhibition