mouse anti human integrin beta 5 itgb5  (Thermo Fisher)

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

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: αvβ5-integrin and chondroitin sulfate proteoglycans are implicated in Wnt1-inducible signaling protein 1 (WISP1)-induced production of migration inhibitory factor (MIF) from human lung fibroblasts (HLFs). HLFs were pretreated for 30 minutes with anti-αvβ5-integrin antibody (6 µg/mL) ( A ) or for 2 h with chondroitinase ABC or chondroitinase AC II (3 units/mL) ( B ), followed by stimulation with WISP1 (100 ng/mL) for 48 h, and the levels of MIF in conditioned media were determined by ELISA. Data represent the means ± SD ( n = 3). *Statistically significant increase ( P < 0.05) compared to control. #Statistically significant decrease ( P < 0.05) compared to WISP1-treated cells. IgG represents immunoglobulins from nonimmunized mouse.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways

doi: 10.1152/ajpcell.00410.2023

Figure Lengend Snippet: Proposed model of the effects of Wnt1-inducible signaling protein 1 (WISP1) on the expression of migration inhibitory factor (MIF) and its receptors, as well as on the expression of cyclooxygenase (COX)-2, IL-6, and matrix metalloproteinase (MMP)-2, and production of prostaglandin E 2 (PGE 2 ) from human lung fibroblasts (HLFs). The effect of WISP-1 on the expression of MIF in HLFs with the complex interactions between WISP-1, αvβ5, Src kinases, EGF receptor (EGFR), and CD74/CD44 receptors, reveal intricate regulatory networks that could contribute to the expression of proinflamamtory molecules and remodeling enzymes secretion.

Article Snippet: Recombinant human EGF and anti-integrin αvβ5 mouse mAb (no. MAB2528-SP) were purchased from R&D Systems (Bio-Techne Ltd., Abingdon, UK).

Techniques: Expressing, Migration

Journal: Cell

Article Title: Type V collagen in scar tissue regulates the size of scar after heart injury

doi: 10.1016/j.cell.2020.06.030

Figure Lengend Snippet: (A) Expression of ECM and myofibroblast genes in Col5a1CKO CFs generated ex vivo (n=6) (B,C) Flow cytometry to determine expression of (B) αvβ3 and (C) αvβ5 integrins on Col5a1CKO CFs (n=6). (D,E) Immunostaining for Vim and (D) αvβ3, (E) αvβ5 in scar tissue at 7 days post-MI (arrows, representative images) (F) Expression of key myofibroblast genes in Col5a1CKO CFs in the presence or absence of cilengitide (n=6). (G) Experimental design to treat animals with daily cilengitide (20mg/kg) (H) EF/FS in control and Col5a1CKO injected with cilengitide or vehicle (*Col5a1CKO+Cilengitide [red dotted line] vs. Col5a1CKO+Veh [red solid line], n=13/CKO+Cilengitide 10/other groups at basal, n=12/CKo+Cilengitide, 6/CKO+Veh, 9/Control+Cilengitide, 7/Control+Veh at 2wks post MI). (I) Representative images of M mode echocardiogram (yellow line indicates end systolic diameter) (J) Masson trichrome staining of mid ventricle at 2 weeks post-MI to show scar size (arrowhead, n=same number at 2 weeks post-MI as above) (K) Quantitation of fibrotic area (n=same number as above) (L) Fraction of Col5a1CKO animals demonstrating mild, moderate and severe fibrosis following PBS or cilengitide infusion (M) Immunostaining for αSMA and Vimentin in hearts of Col5a1CKO receiving PBS or cilengitide and quantitation of the fraction (arrows, representative images, n=10/CKO+Cilengitide, n=6/CKO+Veh, n=6/animals for all other groups). (N) Dot plot representing expression of ECM genes that are upregulated in CFs of Col5a1CKO hearts at 7 days post-MI compared to controls (left panel), the same genes were shown in fibroblasts from Col5a1CKO+Vehicle and Col5a1CKO+Cilengitide samples (right panel). (O) Box plot showing the module scores of 28 genes from (N) in fibroblasts from Col5a1CKO+Veh and Col5a1CKO+Cilengitide. Data shown as mean± S.D., *p<0.05, ns: Not significant.

Article Snippet: Antibodies and probes The following primary antibodies, reagents, or probes were used for immunostaining: rabbit anti-Vimentin (1:100, Abcam, ab45939); mouse anti-smooth muscle actin (1:100, Dako, M0851); anti-cardiac Troponin I (1:100, Abcam, ab47003); mouse anti-integrin αVβ3 (1:50, Abcam, ab7166); mouse integrin αVβ5 (1:20, R&D, MAB2528); Alexa Fluor 594 conjugated WGA (5μg/ml, Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"W11262","term_id":"1285567","term_text":"W11262"}} W11262 ).

Techniques: Expressing, Generated, Ex Vivo, Flow Cytometry, Immunostaining, Control, Injection, Staining, Quantitation Assay