anti human αvβ5 integrin antibody  (R&D Systems)

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 5 MFGE-8 facilitates EV uptake into recipient hESCs through integrin αvβ5. (a,b) The uptake of PKH-labelled EVs (30 µg/mL) that were pre-incubated with different concentrations of rhMFGE-8 (0–50 µg/mL) (a, n = 3) or anti-M8nAb (b, n = 4) for 1 h and then exposed to hESCs. The mean fluorescence intensity of hESC colonies was measured after 6 h. Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. (c) Changes in the uptake of PKH-EVs (30 µg/mL) that were pre-incubated with or without rhMFGE-8 (25 µg/mL) for 1 h and exposed to hESCs for 6 h. anti- M8nAb (5 µg/mL) was added to the hESC culture for 1 h before the cells were exposed to PKH-EVs (n = 4). Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. d, hESCs were exposed to untreated PKH-labelled bare EVs (30 µg/mL) or pre-treated for 3 h with hESC-derived SFs (bare EVs + SFs) or with hESC-derived SFs in which MFGE-8 activity was inhibited by anti-M8nAb (50 µg/mL). The mean fluorescence intensity was measured after 6 h of exposure (n = 4). Cells were stained for OCT4, and nuclei were stained with DAPI. (e) hESCs were exposed to PKH-EVs (30

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Incubation, Fluorescence, Staining, Derivative Assay, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 6 MFGE-8 mediates EV uptake in hESCs through the integrin αvβ5/Akt/GSK3β pathway. (a) A human phospho-kinase antibody array for hESCs cultured for 2 h in the absence or presence of anti-M8nAb (5 µg/mL) to block EV uptake. (b,c) Immunoblot showing phosphorylation of AKT and GSK3β after hESCs were treated with rhMFGE-8 (b) or hESC-derived EVs (c), with relative ratios of phosphorylation of AKT and GSK3β after 1 h of treatment shown on the right (n = 3). (d–f) Immunoblots showing the phosphorylation of AKT and GSK3β after hESCs were treated with different combinations of rhMFGE-8 (5 µg/mL), EVs (50 µg/mL), anti-M8nAb (5 µg/mL), anti-integrin αvβ5 antibody (anti-ITG αvβ5, 10 µg/mL), LY294002 (10 µM) and/or CHIR99021 (2 µM). hESCs were pre-incubated with or without anti-M8nAb, anti-integrin αvβ5 Ab, LY294002 and CHIR99021 for 1 h. Cells were treated with rhMFGE-8 or hESC-EVs for 15 min and 30 min, respectively. Relative phosphorylation is shown as a bar graph on the right (n = 3). (g) Image of PKH-EV uptake in hESCs treated with the factors indicated. PKH-labelled EVs (30 µg/mL) were pre-incubated with or without anti-M8nAb (25 µg/mL) for 1 h and exposed to hESCs for 6 h. The anti-ITG αvβ5 (10 µg/mL) or LY294002 (10 µM) was added to the hESC culture for 1 h before the cells were exposed to the PKH-EVs. Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. Relative EV uptake is shown as a

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Ab Array, Cell Culture, Blocking Assay, Western Blot, Phospho-proteomics, Derivative Assay, Incubation, Staining

Journal: Journal of extracellular vesicles

Article Title: MFGE-8, a Corona Protein on Extracellular Vesicles, Mediates Self-Renewal and Survival of Human Pluripotent Stem Cells.

doi: 10.1002/jev2.70056

Figure Lengend Snippet: FIGURE 7 MFGE-8 promotes endocytosis of EVs and self-renewal of hESCs by activating dynamin-1 and cyclin D1 via the integrin αvβ5/Akt/GSK3β axis. (a) Relative EV uptake in hESC cells treated with endocytosis inhibitors for 1 h (n = 3). hESC cells were incubated with PKH-EVs (30 µg/mL) for 6 h. (b) Images and quantitation of PKH-EV uptake in hESCs. EVs were pre-incubated with or without rhMFGE-8 (25 µg/mL) for 1 h before they were used to treat hESCs in the presence or absence of dynasore (DNS, 10 µM) (n = 4). Cells were stained for OCT4 and E-cadherin, and nuclei were stained with DAPI. (c) RT-qPCR analysis of DNM1 and DNM2 expression in hESCs (n = 3). (d) EV uptake in hESCs after siRNA knockdown of dynamin 1 (siDNM1) and dynamin 2 (siDNM2). Cell nuclei were stained with DAPI. Two siRNAs (#1 and #2) targeting different mRNA sequences of dynamins were used (n = 4). (e) Immunoblots showing phosphorylation of DNM1 in hESCs treated with or without 100 µg/mL hESC-EVs for 1 h. The relative phosphorylation of DNM1, quantitated by densitometry, is shown as a bar graph on the left (n = 3). (f) Images and quantitation of EV uptake for hESCs treated with combinations of anti-M8nAb and inhibitors, as indicated. hESCs were pre-treated with LY294002 (10 µM), CHIR99021 (2 µM), DNS (10 µM) or chlorpromazine (CPZ) (1 µM). hESC-EVs were pre-incubated with or without anti-M8nAb (25 µg/mL) for 1 h before exposure to cells (n = 3). Cells

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (1:400; sc-5279, Santa Cruz Biotechnology), rabbit anti-vimentin (1:200; 5741, Cell Signalling Technology), goat anti-E-cadherin (1:400; AF648, R&D Systems), mouse anti-integrin αvβ5 (1:100; MAB2528, R&D Systems), mouse anti-8-oxoguanine (1:200; MAB3560, Millipore) and rabbit anti-53BP1 (1:200; 4937, Cell Signalling Technology).

Techniques: Incubation, Quantitation Assay, Staining, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Phospho-proteomics