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: To analyze the membrane fraction in Neuro‐2a cells, the samples were prepared using a Minute Plasma Membrane Protein Isolation and Cell Fractionation Kit (SM‐005, Invent Biotechnologies) according to the manufacturer's instructions.

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

Journal: Annals of Medicine

Article Title: MERTK-related engulfment of norepinephrinergic neuronal presynaptic membrane by astrocytes following acute TBI and potentiation effect of peripheral nerve modulation

doi: 10.1080/07853890.2026.2735720

Figure Lengend Snippet: Astrocytes showed activated phagocytic ability in LC following acute TBI. (A and D) The location of LC in control group and TBI group. The irregular dashed line outlining the range of DBH represents the LC. Scale bar: 100 μm. (B and C) Co-labeling of astrocytes and Lamp2 in LC of control group. Co-labeling of GFAP and Lamp2 in LC of control group were shown by the head of white arrows in (C), with the inset in the upper right corner showing an enlarged view of the indicated area. (E and F) Co-labeling of astrocytes and Lamp2 in LC of TBI group. Co-labeling of GFAP and Lamp2 in LC of TBI group were shown by the head of white arrows in (F), with the inset in the upper right corner showing an enlarged view of the indicated area. (B and E) Scale bar: 100 μm. (C and F)Scale bar: 20 μm. (G) Co-labeling of Lamp2 and GFAP in LC is enhanced following TBI, which indicates an increased phagocytic capacity of astrocytes. (each group, n = 5). t-tests employed for (G). (Contol group vs. TBI group, *** p < 0.001). IV:Fourth ventricle.

Article Snippet: The primary antibodies used in this study were: Rabbit anti-Dopamine beta Hydroxylase (DBH) (ab209487, 1:500, Abcam), Rabbit anti-cFos (2250 T, 1:1000, Cell signaling Technology), Rabbit anti-Caspase-3 p12 (A5357, 1:500, Selleck), Mouse anti-Glial Fibrillary Acidic Protein (GFAP) (ab279290, 1:500, Abcam), Rabbit anti-Norepinephrine transporter (NET) (ab254361, 1:100, Abcam), Rabbit anti-Lysosome-associated membrane protein 2 (Lamp2) (bs-2739R, 1:200, Bioss), Rabbit anti-MERTK (ab184086, 1:500, Abcam).

Techniques: Control, Labeling

Journal: Journal of Neuroinflammation

Article Title: Human umbilical cord blood mononuclear cells ameliorate vascular dementia by modulating microglial myelin debris handling and white matter injury

doi: 10.1186/s12974-026-03929-6

Figure Lengend Snippet: hUCB-MNCs treatment reduces myelin debris burden and improves microglial debris-handling responses. A Representative immunofluorescence images showing degraded myelin basic protein (dMBP), Iba1, DAPI, and dMBP signals within Iba1-positive cells in the CC. B – E Quantification of total dMBP volume ( B ), Iba1 volume ( C ), dMBP volume within Iba1-positive cells ( D ), and the percentage of dMBP-positive staining within Iba1-positive cells ( E ). F , G Representative immunofluorescence images of Iba1, BODIPY, and DAPI staining in BV2 microglial cells incubated with myelin debris under control, OGD, and OGD plus hUCB-MNC-conditioned medium conditions ( F ), with quantification of BODIPY fluorescence intensity ( G ). H , I Representative immunofluorescence images of Iba1, LAMP, and DAPI staining in BV2 microglial cells incubated with myelin debris under control, OGD, and OGD plus hUCB-MNC-conditioned medium conditions ( H ), with quantification of LAMP fluorescence intensity ( I ). J , K Representative immunofluorescence images of Iba1, p21, and DAPI co-staining in the CC of Sham, BCAS, and BCAS plus hUCB-MNCs-treated mice ( J ), with quantification of p21 fluorescence intensity and the percentage of p21 ⁺ Iba1 ⁺ overlapping area relative to total Iba1 ⁺ area ( K ). Data are presented as mean ± SD. For in vivo dMBP/Iba1 three-dimensional reconstruction analysis, n = 6 male mice per group, with each data point representing one mouse. For in vitro Boron-Dipyrromethene (BODIPY) and Lysosome-Associated Membrane Protein (LAMP) immunofluorescence analyses, n = 4 independent experiments, with each data point representing one independent experiment. For in vivo p21 staining analysis, n = 5 male mice per group, with each data point representing one mouse. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars are indicated in the images

Article Snippet: After blocking, cells were incubated with the indicated primary antibodies, including anti-Iba1 (Rabbit anti-Iba1, 1:1000, Wako, Japan, 019-19741; Guinea pig anti-Iba1, 1:500, Oasis, China, OB-PGP049), anti-MBP (Rabbit anti-MBP, 1:200, Proteintech, China, 10458-1-AP) and anti- Lysosome-Associated Membrane Protein (LAMP) (1:200, CST, USA, 99437) antibodies, overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Incubation, Control, Fluorescence, In Vivo, Analysis, In Vitro, Membrane