Fig 1: (a–f) CD63-immunoreactivity in control and 7-day-axotomized motor neurons (MNs). (a) A control MN showing CD63-immunolabeling in the form of abundant particles (green); no association is found with VAChT-positive synaptic terminals (blue). (b) After axotomy, many CD63-positive particles (green) are seen interacting with microglial cells (Iba1-immnunolabeled, red) recruited at the MN surface; the boxed are is shown enlarged in (c). (d–f) Pixel intensity (arbitrary units [a.u.]) profile along a line (yellow) traced along the periphery of the control and axotomized MN cell bodies, showing the increase in CD63-positive particles postaxotomy. (g–l) Flotillin-immunoreactivity in control and 7-day-axotomized MNs. (g) A control MN showing flotillin-immunolabeling in the form of abundant particles (green); no association is found with VAChT-positive synaptic terminals (blue). (h) After axotomy, many flotillin-positive profiles (green) are seen in close proximity to microglial cells (Iba1-immnunolabeled, red) recruited at the MN surface; the boxed area is shown enlarged in (i). (j–l) Pixel intensity (arbitrary units [a.u.]) profile along a line (yellow) traced along the periphery of the control and axotomized MN cell bodies, showing the increase in flotillin-positive particles postaxotomy. Scale bars: e = 10 µm (valid for a, b, d); c = 5 µm; k = 15 µm (valid for g, h, j); and i = 5 µm [Color figure can be viewed at wileyonlinelibrary.com]
Fig 2: Cocaine injection does not affect the level of expression of EV protein markers and of a-synuclein. a–h Western blot analyses of brain homogenates of female (a), male (b), and females undergone sham (e) or ovariectomy (f) surgeries injected with either saline (S) or cocaine (C) (n = 7 mice for the female set, 6 for the other groups). The antibodies detected a marker of microvesicles (Annexin A2), markers of exosomes (Alix, Tsg101, Hsc70, Cd63, Rab27a, and Rab35), and a-synuclein. ß-actin was used as a loading control. The same amount of total protein was loaded in each lane. Graphs in c, d, g and h show the densitometric quantification of the bands normalized to ß-actin in the same lane. Statistical test: two-way ANOVA with Bonferroni’s multiple comparisons test. No difference between cocaine and saline treatments was found. Data are represented as mean ± SEM
Fig 3: Characterizations of UC-based nanoparticles. A TEM and DLS analysis of UC-based nanoparticles. B EDX mapping of EMS. C Immunogold labeling of CD63 of EMS. D Cumulative release of SAHA from EMS in different pHs. The data in D are expressed as mean ± SD, n = 3. The comparison was performed with unpaired two-way Student’s t-tests. *P < 0.05
Fig 4: Exosome characterization and internalization pathways. (A) Transmission electron microscopy images of exosomes isolated from the two groups. Scale bar, 100 nm. (B) Protein expression of the exosomal markers Alix, CD63, and TSG101. (C) Exosome particle diameter distribution. (D) The concentration of exosome between control group and AMI individuals. (E) Exosome uptake was analyzed at the indicated times using flow cytometry. (F) Exosome uptake in human umbilical vein endothelial cells (HUVECs) incubated with PKH67-labeled exosomes (green), as analyzed by confocal imaging. Nuclei were dyed with DAPI (blue). Scale bar, 20 µm.
Fig 5: Characteristics and expression of circ-0004277 in HCC. (A) The expression level of six well-known circRNAs was detected in the normal human hepatic cell line HL-7702 and HCC cell lines through qRT-PCR. (B) qRT-PCR detection of the relative expression of circ-0004277 in paired HCC tumor and normal tissues (n = 60). (C) The sequence of circ-0004277 in circBase (upper panel) showed consistent results with Sanger sequencing (lower panel). (D) Circ-0004277 was resistant to RNaseR digestion in HCC cell lines. (E) Micrographs of plasma-derived exosomes in negative controls (left) and HCC patients (right, bars = 200 nm). (F) Western blots of CD63 and TSG101 in circulating exosomes. (G) qRT-PCR of circ-0004277 in plasma-derived exosomes. (H) ROC curve of exosomal circ-0004277 signature. Results are reported as mean ± SD. *P < 0.05, ***P < 0.001. All experiments were performed in triplicate.
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