Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Olfactory mucosa mesenchymal stem cell‐derived exosomes protect against neuroinflammation after subarachnoid hemorrhage by activating mitophagy

doi: 10.1002/kjm2.12951

Figure Lengend Snippet: Identification of OM‐MSCs and exosomes. (A) Cell morphology was observed under a microscope. (B) Vimentin and Nestin levels were determined using immunofluorescence staining. (C) The expression of CD44, CD73, CD34, and CD45 was detected by flow cytometry. (D) Transmission electron microscopy was used to observe the morphology of the exosomes. (E) The peak particle size of the purified exosomes was assessed by NTA. (F) Western blot detection of CD81, TSG101, and Calnexin levels in purified exosomes. (G) Fluorescent PKH26‐labeled exosomes taken up by HT22 cells were observed via fluorescence microscopy. n = 3.

Article Snippet: Calnexin (10427‐2‐AP, 1:10000, Proteintech), TSG101 (28283‐1‐AP, 1:5000, Proteintech), and CD81 (27855‐1‐AP, 1:1000, Proteintech) levels were verified by Western blotting.

Techniques: Microscopy, Immunofluorescence, Staining, Expressing, Flow Cytometry, Transmission Assay, Electron Microscopy, Purification, Western Blot, Labeling, Fluorescence

Journal: Frontiers in Immunology

Article Title: R3HDM4 influences kidney renal clear cell carcinoma progression, immune modulation, and potential links to the IGSF8 immune checkpoint

doi: 10.3389/fimmu.2025.1722358

Figure Lengend Snippet: R3HDM4 silencing modulates expression of EMT, invasion, and immune-related markers in 786-O cells. (A) Representative Western blot of E-cadherin, MMP-2, MMP-9, Vimentin, IGSF8 and GAPDH in 786-O (control), 786-O+si-NC (negative control siRNA), and 786-O+si-R3HDM4-4 (R3HDM4-targeting siRNA) groups. (B) Quantification of relative E-cadherin expression across 786-O, si-NC, and si-R3HDM4–4 groups. (C) Quantification of relative MMP-2 expression across 786-O, si-NC, and si-R3HDM4–4 groups. (D) Quantification of relative MMP-9 expression across 786-O, si-NC, and si-R3HDM4–4 groups. (E) Quantification of relative Vimentin expression across 786-O, si-NC, and si-R3HDM4–4 groups. (F) Quantification of relative IGSF8 expression across 786-O, si-NC, and si-R3HDM4–4 groups. P - values were determined by one-way ANOVA; ** P < 0.001 indicates statistical significance.* P < 0.05, ** P < 0.01, *** P < 0.001. CTRL, control untreated; si-NC, negative control siRNA; si-R3HDM4, R3HDM4-targeting siRNA; E-cadherin, epithelial cadherin; MMP-2/9, matrix metalloproteinase-2/9; IGSF8, Immunoglobulin Superfamily Member 8 expression.

Article Snippet: The primary antibodies used were: R3HDM4 (29 kDa, 1:1000, Invitrogen, USA), GAPDH (36 kDa, 1:10,000, Proteintech, China), E-cadherin (125 kDa, 1:40,000, Proteintech, China), Vimentin (55 kDa, 1:40,000, Proteintech, China), MMP2 (63 kDa, 1:1000, BIOSS, China), MMP9 (78 kDa, 1:1000, Affinity, USA), and IGSF8 (70 kDa, 1:2000, Proteintech, China).

Techniques: Expressing, Western Blot, Control, Negative Control

Journal: Cells

Article Title: Calmodulin as a Key Regulator of Exosomal Signal Peptides

doi: 10.3390/cells12010158

Figure Lengend Snippet: Interaction of CaM with HSP70 and CD81 in T-REx cells ( A ) The peptide solution extracted from immunoprecipitants with the CaM antibody from T-REx cells was analyzed by MALDI-TOF-MS. ( B ) The peak intensity at m/z 861 was measured by MALDI-TOF-MS. ** p < 0.01 vs. Mock cells in the absence of W13 and EGTA. C; control, Mock; T-REx Mock, AspALP; T-REx AspALP. ( C ) Western blot analysis of immunoprecipitants with the CaM antibody from T-REx cells. Representative images from three independent experiments.

Article Snippet: Primary antibodies were directed against CD81 (Novus Biologicals, Centennial, CO) and CD63 (Novus Biologicals) under non-reducing conditions and against CaM (Abcam, Cambridge, UK), Alix (Cell Signaling Technology), HSP70 (Enzo Life Sciences, Farmingdale, NY, USA), and GAPDH (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan).

Techniques: Control, Western Blot

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: Flow cytometric characterization of CD81 and LDLr expression on MOLT-4 cells. MOLT-4 cells were grown for 48 h in lipoprotein-rich medium (A) or for 24 h (B) or 48 h (C) in lipoprotein-deficient medium. Background fluorescence was measured with an irrelevant isotype-matched control MAb. CD81 expression was measured with JS64 MAb, and LDLr expression was measured with C7 MAb. Cell-bound antibodies were detected with anti-mouse IgG Oregon green (FITC).

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Expressing, Fluorescence

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: HCV and E2 binding to MOLT-4 cells with low or high levels of LDLr expression

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Binding Assay, Expressing

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: Inhibition of HCV and HCV E2 protein binding to MOLT-4 cells by human soluble CD81, mouse soluble CD81, and LDL

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Inhibition, Protein Binding

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: Specificity of HCV-E2 interactions with human CD81. Soluble human or mouse CD81 (10 μg/ml) applied to nitrocellulose was incubated with HCV-E2 (10 μg/ml). E2 applied to nitrocellulose served as the positive control, and an irrelevant control peptide served as the negative control. Interactions of E2 with each protein were detected with anti HCV-E2 MAb, anti-mouse IgG-AP, and 5-bromo-4-chloro-3-indolyl phosphate–nitroblue tetrazolium substrate.

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Incubation, Positive Control, Negative Control

Journal:

Article Title: Characterization of Hepatitis C Virus (HCV) and HCV E2 Interactions with CD81 and the Low-Density Lipoprotein Receptor

doi:

Figure Lengend Snippet: HCV-E2 does not interact with LDL. HCV-E2, HCV NS3/4, human soluble CD81, or mouse soluble CD81 spotted on nitrocellulose were incubated with LDL (10 μg/ml). The positive control consisted of goat-anti LDL polyclonal antibody spotted on nitrocellulose. Interaction of spotted proteins with LDL was detected with mouse anti-LDL MAb and anti-mouse IgG AP followed by 5-bromo-4-chloro-3-indolyl phosphate–nitroblue tetrazolium substrate. Binding was quantitated by densitometry (AlphaImager 2000; Alpha Innotech Corp., San Leandro, Calif.), and results represent density units × 100.

Article Snippet: Anti-human CD81 MAb (clone JS64) was obtained from RD Inc. (Flanders, NJ).

Techniques: Incubation, Positive Control, Binding Assay

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: Characterization of EV. Particle numbers (A) and relative protein concentrations (B) in the fractions eluted following qEV size-exclusion chromatography. Data are mean ± SEM. (C) Tetraspanin (CD9, CD63, and CD81) content of purified EVs was analyzed in the indicated elution fractions by immunoblotting. (D) Size distribution of pooled fractions 7 to 10 from HDFs (left panel) and L3.6 cells (right panel). (E) Electron microscopy image of EV from HDFs (left panel) and L3.6 cells (right panel).

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Size-exclusion Chromatography, Purification, Western Blot, Electron Microscopy

Journal: Blood Advances

Article Title: Polyphosphate expression by cancer cell extracellular vesicles mediates binding of factor XII and contact activation

doi: 10.1182/bloodadvances.2021005116

Figure Lengend Snippet: polyP on EV derived from cancer cells mediates ligand binding. (A) The amount of polyP associated with cancer cell–derived EV was estimated by comparison with a standard curve (supplemental Figure 2A). (B) Detection of polyP on L3.6-derived EVs following incubation with buffer (HBS), DNase I and RNase A (D/R), or CIP. polyP of different chain lengths was used to estimate the size of EV-associated polyP. L3.6-derived EVs were preincubated with buffer or E coli PPX, and the binding of PPX-Δ12–AF488 (C), anti-CD81–AF488 (D), and or FXII-AF488 (E) was measured. The number of total EV was determined in scatter mode, and the number of ligand-binding EV was determined in fluorescence mode using a 488-nm excitation laser. The differences in fluorescent particle numbers were compared by estimating the area under each curve (AUC) using GraphPad Prism 8. Bars represent means ± SEM. *** P < .001, 1-way ANOVA with multiple comparisons. L, long; M, medium; S, short.

Article Snippet: Anti-human CD81-AF488 antibody (20 μg/mL, FAB4615G; R&D Systems) was used as a control ligand.

Techniques: Derivative Assay, Ligand Binding Assay, Comparison, Incubation, Binding Assay, Fluorescence