Journal: Frontiers in Immunology

Article Title: Extracellular vesicles derived from antigen-presenting cells pulsed with foot and mouth virus vaccine-antigens act as carriers of viral proteins and stimulate B cell response

doi: 10.3389/fimmu.2024.1440667

Figure Lengend Snippet: EVs-FMDVi express FMDV proteins, classical EVs markers, and immunoregulatory molecules. EVs were coupled with aldehyde/sulfate latex beads of 4 µm diameter and incubated with different fluorochrome-conjugated monoclonal antibodies anti-CD9, anti-CD81, CD63, MHC-II, CD11c, CD86 as detailed in materials and methods. For viral protein detection on EVs, FITC-labeled IgG purified from an FMDV-immune bovine serum was used, and FITC-labeled IgG obtained from a healthy unimmunized bovine serum was used as a corresponding control (Control). Comparison of percentages for different marker proteins and FMDV viral proteins for each EV population. The mean value ± SEM of the Median Fluorescence Intensity (MFI) for each of the markers evaluated is also shown. For statistical analysis, the Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used for the analysis of MFI, and one-factor ANOVA followed by Bonferroni’s multiple comparisons test for the analysis of percentages. Only significant differences are indicated in the graphs (p<0.05 *; p<0.01**; p<0.001 ***). The data correspond to 15 independent experiments.

Article Snippet: After being saturated with 100 mM glycine, EVs were incubated with the following monoclonal antibodies: 0.02µg/100µl anti-MHC-II-PE (Biolegend-clone: M5/114.15.2), 0.2µg/100µl anti-CD11c-APC (Biolegend-clone: N418), 1.5 µg/100µl anti-CD86-FITC (Miltenyi-clone: PO3.3), 1 µg/100µl anti-CD9-biotin (eBioscience-clone: MEM61), 1 µg/100µl anti-CD81-biotin (eBioscience clone: Eat2) or 1 µg/100µl polyclonal antibody anti-CD63-biotin (MyBioSource).

Techniques: Incubation, Labeling, Purification, Control, Comparison, Marker, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

doi: 10.1074/jbc.M117.779710

Figure Lengend Snippet: TG-A is secreted in exosomes via an unconventional secretion pathway. A and D, brefeldin A (1 μg/ml, B) or monensin (1 μm, M) were added to the C-terminal FLAG-tagged TG-A, TG-B, AN46-EGFP, or BN38-EGFP-expressing S2 cells, and subsequently treated with A23187 or ionomycin (1 μm each). Cell lysates and the P100 fraction were analyzed by Western blotting using the anti-TG-A/B (A) or anti-GFP tag antibody (D). B, GFP fused with the BiP secretion signal sequence at the N terminus was expressed in S2 cells and treated with brefeldin A (1 μg/ml) or monensin (1 μm). The resulting cultured medium was analyzed by Western blotting using the anti-GFP tag antibody. C, A23187 or ionomycin (1 μm each) were added to the C-terminal EGFP-tagged N-terminal fragment of TG-A and TG-B (AN46-EGFP, BN38-EGFP)-expressing S2 cells for 1 h, and analyzed by Western blotting using the anti-GFP tag antibody. E, the C-terminal EGFP-tagged TG-A-expressing S2 cells were treated with or without 10 μm GW4869 for 40 h, and then with or without 1 μm A23187 for 1 h. Bar graph shows the band intensity analyzed by ImageJ software, and error bars indicate ± S.E. (n = 3). F, the P100 fraction from dsRab27 or dsEGFP (negative control)-treated C-terminal V5-His6-tagged TG-A-expressing S2 cells were analyzed by Western blotting using the anti-His6 tag antibody. Bar graph shows the band intensity analyzed by ImageJ software, and error bars indicate ± S.E. (n = 6). G, the P100 fraction from the C-terminal FLAG-tagged TG-A and/or the C-terminal EGFP-tagged human CD63-expressing S2 cells were analyzed by Western blotting using the anti-FLAG, CD63, or GFP antibody.

Article Snippet: The blocked membrane was reacted to each antibody at room temperature for 1 h, including the anti-TG/B polyclonal antibody, anti-His tag mAb-HRP-DirecT (catalog number D291-7, MBL), anti-V5 tag HRP-DirecT (catalog number PM003-7, MBL), anti-DDDDK tag mAb-HRP-DirecT (catalog number M185-7, MBL), anti-GFP-HRP (catalog number 120-002-105, Miltenyi Biotec, Bergisch Gladbach, Germany), or anti-CD63 monoclonal antibody TS63 (catalog number 10628D, Life Technologies).

Techniques: Expressing, Western Blot, Sequencing, Cell Culture, Software, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations

doi: 10.1074/jbc.M117.779710

Figure Lengend Snippet: Profiles of TG-A containing exosomes. A, the P100 fraction prepared from A23187-stimulated the C-terminal FLAG tagged-TG-A- and the N-terminal EGFP-tagged-CD63-expressing S2 cells was analyzed by density gradient centrifugation using OptiPrep. B and C, the P100 fraction from A23187-stimulated the C-terminal FLAG tagged-TG-A-expressing S2 cells was analyzed by the biotin-switch assay with or without HA (B), or treated with or without 10 μm proteinase K in the presence or absence of 1% Triton X-100 for 1 h at 37 °C (C). D, immunotransmission electron microscopic analysis of S2 cells expressing the C-terminal FLAG-tagged TG-A. The TG-A-specific antibody was used as a primary antibody and was detected by the anti-rabbit colloidal-gold conjugated secondary antibody. a′ and b′ represent magnified photographs of square area of a and b, respectively. Arrowheads indicate 10-nm colloidal gold signals. The scale bars in white in a and b, or a′ and b′ are 500 and 100 nm, respectively. E, intracellular gold particles from 10 cells in the immune transmission electron microscopic analysis were counted and analyzed by Student's t test. ***, p < 0.001. Error bars indicate ± S.E. (n = 10). F, the P100 fraction prepared from A23187-treated (1 or 2 μm) the C-terminal V5-His6-tagged TG-A-expressing S2 cells were incubated with Ecc15 for 1 h at room temperature. After incubation, bacteria were collected and washed three times, and then bacteria-bound proteins were analyzed by Western blotting using the anti-His6 tag antibody. Numbers show the band intensity of the bacteria-bound fraction analyzed by ImageJ software, and the intensity at the 2 μm A23187 was set to 1.0. ND, not detectable. G, S2 cells expressing the C-terminal EGFP tagged-TG-A were labeled with the exosomal marker BODIPY TR ceramide, and stimulated with A23187, and then the resulting P100 fraction was collected and added to untransfected S2 cells for 1 h. The exosome-treated S2 cells were analyzed under a fluorescence microscope. The scale bars in white are 10 μm.

Article Snippet: The blocked membrane was reacted to each antibody at room temperature for 1 h, including the anti-TG/B polyclonal antibody, anti-His tag mAb-HRP-DirecT (catalog number D291-7, MBL), anti-V5 tag HRP-DirecT (catalog number PM003-7, MBL), anti-DDDDK tag mAb-HRP-DirecT (catalog number M185-7, MBL), anti-GFP-HRP (catalog number 120-002-105, Miltenyi Biotec, Bergisch Gladbach, Germany), or anti-CD63 monoclonal antibody TS63 (catalog number 10628D, Life Technologies).

Techniques: Expressing, Gradient Centrifugation, Biotin Switch Assay, Transmission Assay, Incubation, Western Blot, Software, Labeling, Marker, Fluorescence, Microscopy