polyclonal anti–dpp-4 antibody  (Santa Cruz Biotechnology)

Journal: Aging (Albany NY)

Article Title: New insights into ginsenoside Rg1 regulating the niche to inhibit age-induced germline stem cells depletion through targeting ECR/BMP signaling pathway in Drosophila

doi: 10.18632/aging.205548

Figure Lengend Snippet: Effect of ginsenoside Rg1 on niche ECR/BMP signaling in Drosophila . ( A ) Western blotting analysis of expression of ISWI, Decapentaplegic (Dpp) and Smad4; ( B ) Relative expression levels of ISWI; ( C ) Relative expression levels of Dpp; ( D ) Relative expression levels of Smad4; ( E ) Western blotting analysis of expression of pSmad1/5/8; ( F ) Relative pSmad1/5/8 expression levels; GAPDH antibody was used as loading Control. Results were analysed with one-way ANOVA. Data are shown as the mean ± SD ( n = 100); ### p < 0.001 compared with Control (7-day-old female Drosophila ); *** p < 0.001 compared with Model (35-day-old female Drosophila ); +++ p < 0.001 compared with Control (7-day-old female Drosophila ); Abbreviation: ns: no significance.

Article Snippet: After blocked with 5% non-fat milk, PVD membranes were immunoblotted with the following antibodies: ISWI (1:1000, ab 195309), Nos (2 μg/ml, ab 70001) (both from Abcam, USA); Smad1/5/8 (#12656), pSmad1/5/8 (#13820) (1:500, both from Cell Signaling Technology, USA); Decapentaplegic (Dpp) (1:100, sc-133182), Smad4 (1:200, sc-7966) (both from Santa Cruz Biotechnology, USA), and Bam (3 μg/ml, DSHB, NIH, USA).

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Flow chart of procedure for isolating salivary extracellular vesicles. Salivary extracellular vesicles (EVs) were isolated using a combination of size-exclusion chromatography and sequential centrifugation. Human whole saliva was centrifuged, and the supernatant was filtrated and then, concentrated using Amicon Ultra-15. The resulting concentrated solution was applied to a size-exclusion column, and the fractions (Fr.) of EV-I and Fr. EV-II were determined from the measurement of OD280, DPP IV activity, and APN activity. Each fraction was collected, filtered, and concentrated by Amicon Ultra-4 to obtain Fr. EV-I and Fr. EV-II. Each pooled fraction was subjected to sequential centrifugation. The precipitate obtained by centrifugation at 20,000 × g (EV-I or EV-II 20 k-ppt) and the precipitate obtained by centrifugation at 100,000 × g (EV-I or EV-II 100 k-ppt) were further analyzed. After characterizing the protein components (SDS-PAGE and western blotting) and morphological analyses (TEM and DLS), we focused on EV-I 20 k-ppt and EV-II 100 k-ppt and subjected them to proteomic analyses.

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: Isolation, Size-exclusion Chromatography, Centrifugation, Activity Assay, SDS Page, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Preparation of EVs derived from human whole saliva. (upper) Size-exclusion chromatography (Sephacryl S-1000 SF) elution profiles of the EVs from fresh human WS (donor A). The filtrated and concentrated WS (1.5–2.0 mL) was subjected to size-exclusion chromatography on a Sephacryl S-1000 SF column (1.5 cm × 50 cm, 0.33 mL/min) equilibrated with Tris-buffered saline (20 mM Tris-HCl, pH 7.4, and 150 mM NaCl), and 80 fractions (1.2 mL/fraction) were collected within 4 h. All fractions were analyzed for DPP IV and APN activities, and absorbance at 280 nm. The experiments were performed at least three times for each donor, and a representative elution pattern is shown. (lower) Western blot analysis of proteins located on the outer surface (MUC5B and IgA), membrane (MUC1, APN, DPP IV, and CD9), and inner lumen (Alix, TSG101, ezrin, and Annexin A1) of the salivary EV fractions eluted from size-exclusion columns (donor A). Numbers refer to the different fractions obtained via size-exclusion column chromatography shown in the upper panel. Overall, 20 µL of each EV fraction was subjected to SDS-PAGE and analyzed using western blotting. The elution profiles and western blotting of EVs from other donors are shown in .

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: Derivative Assay, Size-exclusion Chromatography, Saline, Western Blot, Membrane, Column Chromatography, SDS Page

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Sequential centrifugation of two types of salivary EVs. Salivary Fr. EV-I and Fr. EV-II were subjected to sequential centrifugation . (A) From each fraction, samples of 2 µg of protein were subjected to SDS-PAGE and silver staining (donor A) was performed to visualize the bands. The silver staining of EVs from other donors is shown in . Grey arrowheads, protein bands shared by Fr. EV-I and Fr. EV-II; black arrowhead, observed predominantly in Fr. EV-I; open arrowheads, observed predominantly in Fr. EV-II. (B) Western blot analysis of proteins located on the outer surface (MUC5B and IgA), membrane (MUC1, APN, DPP IV, and CD9), and inner lumen (Alix, TSG101, ezrin, and Annexin A1) of salivary EVs (donor A). From each EV fraction, samples of 2 µg of protein were subjected to SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with antibodies. Western blots of EVs from other donors are shown in . (C) Morphological analysis of salivary EV fractions visualized using an electron microscope (donor A). Scale bar, 100 nm. The morphology of EVs from other donors is shown in . (D) Particle sizes of salivary EVs analyzed via dynamic light scattering (DLS) measurements conducted in triplicate. A typical result is shown (donor A). The mean particle size of EV-I 20 k-ppt was 263 ± 30 nm (7–14 experiments) and that of EV-II 100 k-ppt was 36.5 ± 13 nm (three to six experiments) .

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: Centrifugation, SDS Page, Silver Staining, Western Blot, Membrane, Microscopy

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Isolation profiles of salivary EVs from Fr. EV-I or Fr. EV-II by sequential centrifugation.

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: Isolation, Centrifugation, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Characteristic proteins in salivary EVs.

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: MUC1, DPP IV, and CD9 were expressed on the surface of salivary EVs. Immunoprecipitation of salivary EVs. Salivary EVs (Fr. EV-I or Fr. EV-II) were immunoprecipitated using magnetic beads. Antibody-coupled beads were added to 5 μg of EVs and the mixture was incubated at 4°C for 18 h on a rotator. The proteins eluted from the beads were subjected to SDS-PAGE. Western blots of EVs from other donors are shown in . Immunoprecipitation with an anti-APN antibody is shown in .

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: Immunoprecipitation, Magnetic Beads, Incubation, SDS Page, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Sequential immunoprecipitation of salivary EVs. (A) Flow chart of procedure for sequential immunoprecipitation. Two types of salivary EVs were sequentially immunoprecipitated from the WS using magnetic beads. Pretreated WS (1 mL) was incubated with anti-DPP IV antibody-coupled beads at 4°C for 3 h on a rotator (first immunoprecipitation). After separating the beads, the WS was further incubated with anti-MUC1 antibody-coupled beads (second immunoprecipitation) at 4°C for 18 h. For the control experiment, nonspecific goat IgG-conjugated magnetic beads and nonspecific mouse IgG-conjugated beads were used for the first and second immunoprecipitations, respectively. The immunoprecipitated beads were washed with PBS, and then sample buffer (20 μL) was added and the solution was denatured at 70°C for 10 min. The eluates were then subjected to western blot analysis. (B) Western blot analysis of the proteins located on the membrane (MUC1, APN, DPP IV and CD9) and inner lumen (Alix and ezrin) of the salivary EVs (donor A). Western blots of EVs from other donors are shown in .

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: Immunoprecipitation, Magnetic Beads, Incubation, Control, Western Blot, Membrane

Journal: Frontiers in Molecular Biosciences

Article Title: Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles

doi: 10.3389/fmolb.2024.1278955

Figure Lengend Snippet: Schematic model of the components of salivary EVs (EV-I and EV-II). Two distinct populations of EVs are present in human WS: large-sized, APN/MUC1-rich, m/l/EVs/microvesicles (EV-I), and small-sized DPP IV/CD9-rich vesicles containing several sEV components (EV-II).

Article Snippet: Plates were then washed three times with Tris-buffered saline (TBS) with 0.05% Tween 20 (TTBS) and incubated with 50 μL of mouse anti-APN antibody (Santa Cruz Biotechnology, Inc.) for EV-I or goat anti-DPP IV antibody (R&D Systems, Inc.) for EV-II diluted 1:1,000 with 1% BSA–PBS overnight at 4°C.

Techniques: