Af938

Manufactured by R&D Systems

Sourced in United States

AF938 is a sterile, lyophilized preparation of Human Activin A. Activin A is a member of the transforming growth factor beta (TGF-β) superfamily of proteins.

Automatically generated - may contain errors

Lab products found in correlation

Sc 9989, by Santa Cruz Biotechnology (2 mentions) Duolink in situ red starter kit mouse rabbit, by Merck Group (2 mentions) V4139, by Merck Group (2 mentions) Laser point scanning microscope 880, by Zeiss (2 mentions) A21447, by Thermo Fisher Scientific (1 mentions) Ab150361, by Abcam (1 mentions) Plan apochromat 63x na 1.40 oil dic, by Zeiss (1 mentions) Matrigel, by BD (1 mentions) Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (1 mentions) Ficoll paque premium sterile solution, by GE Healthcare (1 mentions) Af1738, by Beyotime (1 mentions) Accutase, by STEMCELL (1 mentions) Ab16288, by Abcam (1 mentions) Ab109250, by Abcam (1 mentions) Ab134053, by Abcam (1 mentions) Ab200834, by Abcam (1 mentions) Ab16285, by Abcam (1 mentions) Y 27632, by STEMCELL (1 mentions) Mtesr medium, by STEMCELL (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)

9 protocols using af938

Vinculin-VE-Cadherin Interaction at Adherens Junctions

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Confluent HUVECs seeded on 24-well plates were subjected to the scratch-wound assay and then processed for PLA using the Duolink In Situ Red Mouse/Rabbit Starter Kit (DUO92101-1KT, Sigma-Aldrich) as described by the manufacturer’s protocol. To probe interactions between vinculin and VE-cadherin, cells were incubated with an anti-vinculin antibody raised in rabbit (V4139, Sigma-Aldrich) and an anti-VE-cadherin antibody raised in mouse (sc-9989, Santa Cruz Biotechnologies). In parallel, cells were also incubated with an anti-VE-cadherin antibody raised in goat (AF938, R and D Systems) and subsequently with an anti-Goat fluorescent-conjugated secondary antibody (A21447, Thermo Fisher Scientific) to label adherens junctions. To quantify co-localization of PLA signal at adherens junctions, high-resolution Z-stack images at multiple positions on the wound edge were acquired on a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with the Zen black software with a Plan Apochromat 63x NA 1.40 oil DIC M27 objective. Briefly, PLA dots were quantified only at adherens junctions, using a similar approach to the co-localization studies described in the section ‘Immunostaining co-localization analysis’, using the VE-cadherin immunofluorescence staining to detect overlapping pixels between junctions and PLA signals.

Carvalho J.R., Fortunato I.C., Fonseca C.G., Pezzarossa A., Barbacena P., Dominguez-Cejudo M.A., Vasconcelos F.F., Santos N.C., Carvalho F.A, & Franco C.A. (2019). Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration. eLife, 8, e45853.

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Protein-Protein Interaction in Endothelial Cells

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After flow microfluidic experiments, HUVECs were processed for PLA using the Duolink In Situ Red Mouse/Rabbit Starter Kit (Sigma-Aldrich, DUO92101-1KT) as described by the manufacturer’s protocol. To probe interactions between VINCULIN and VE-cadherin, cells were incubated with an anti-vinculin antibody raised in rabbit (Sigma-Aldrich, V4139) and an anti-VE-cadherin antibody raised in mouse (Santa Cruz Biotechnologies, sc-9989). In parallel, cells were also incubated with an anti-VE-cadherin antibody raised in goat (R&D Systems, AF938) and subsequently with an anti-goat Alexa 647 secondary antibody (Thermo Fisher Scientific, A21447) to label adherens junctions. To probe interactions between VINCULIN and ITGA5, cells were incubated with an anti-vinculin antibody raised in mouse (Sigma-Aldrich, V9264) and an anti-ITGA5 antibody raised in rabbit (Abcam, ab150361).
To quantify colocalization of PLA signal at adherens junctions, high-resolution Z-stack images at multiple positions were acquired on a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with the Zen black software with a Plan Apochromat 63x NA 1.40 oil DIC M27 objective. Briefly, PLA dots were quantified using ImageJ’ particle analysis tool and the data normalized by the number of cells.

Barbacena P., Dominguez-Cejudo M., Fonseca C.G., Gómez-González M., Faure L.M., Zarkada G., Pena A., Pezzarossa A., Ramalho D., Giarratano Y., Ouarné M., Barata D., Fortunato I.C., Misikova L.H., Mauldin I., Carvalho Y., Trepat X., Roca-Cusachs P., Eichmann A., Bernabeu M.O, & Franco C.A. (2022). Competition for endothelial cell polarity drives vascular morphogenesis in the mouse retina. Developmental Cell, 57(19), 2321-2333.e9.

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Antibody Profiling for Stem Cell Characterization

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The following antibodies were used: goat antibody against human VE-cadherin (1:1,000, AF938; R&D systems); rabbit antibody against human GCN2 (1:1,000; ab134053; Abcam); rabbit antibody against human NANOG (1:500; ab109250; Abcam); rabbit antibody against human OCT4 (1:500; ab200834; Abcam); mouse antibody against human SSEA-1 (1:500; ab16285; Abcam); mouse antibody against human TRA-1 (1:500; ab16288; Abcam); rabbit antibody against phospho-Akt (Ser473) (1:1,000; 31957; Cell Signaling); rabbit antibody against Akt (pan) (1:2,000; 4691; Cell Signaling); Ki67 rabbit monoclonal antibody (1:200; AF1738, Beyotime); Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:1,000; R37118; Life Technologies); Ficoll-Paque PREMIUM sterile solution (17544202; GE Healthcare); CytoTune-iPS 2.0 Sendai Reprogramming Kit (A16517; Thermo Fisher Scientific); Matrigel (354277; BD); mTeSR medium (05850; STEMCELL Technologies); Y-27632 (STEMCELL Technologies); Accutase (07920; STEMCELL Technologies); MTT for cell proliferation detection (C0009S; Beyotime).

Ma B., Li T., Li W., Yang H., Zeng Q., Pan Z., Wang K., Chen Q., Xiong C, & Zhou Z. (2022). Patient-specific and gene-corrected induced pluripotent stem cell-derived endothelial cells elucidate single-cell phenotype of pulmonary veno-occlusive disease. Stem Cell Reports, 17(12), 2674-2689.

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Multiplex RNA and Protein Detection in Meningioma

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The RNAScope Multiplex Fluorescent V2 assay (32310, ACDBio) was performed according to the manufacturer’s protocol. Briefly, 5μm FFPE meningioma sections were incubated with hydrogen peroxide to inhibit endogenous peroxidase, followed by processing for target retrieval and treatment with ProteasePlus. Meningioma sections were subsequently incubated with RNA probes for NOTCH3 (558991-C2, Hs-NOTCH3-C2) and NF2 (1037481-C, Hs-NF2-C1), followed by revelation and amplification steps. Meningioma sections were blocked (5% normal donkey serum, 1X Animal Free blocking, 0.3% Triton X-100) for 1 hour at room temperature and incubated with primary antibody against VE-cadherin (AF938, R&D Systems, 1:100) overnight at 4°C. The next day, meningioma sections were incubated with secondary antibodies for 1 hour at room temperature and counterstained with DAPI (62248, ThermoFisher Scientific). Meningioma sections were imaged on a LSM 800 confocal laser scanning microscope with Airyscan (Zeiss) and analyzed using ImageJ.

Choudhury A., Cady M.A., Lucas C.H., Najem H., Phillips J.J., Palikuqi B., Zakimi N., Joseph T., Birrueta J.O., Chen W.C., Bush N.A., Hervey-Jumper S.L., Klein O.D., Toedebusch C.M., Horbinski C.M., Magill S.T., Bhaduri A., Perry A., Dickinson P.J., Heimberger A.B., Ashworth A., Crouch E.E, & Raleigh D.R. (2023). NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy. bioRxiv.

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Immunostaining of Endothelial Cell Junctions

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HUVECs were plated in 6-well plates coated with 2μg/mL fibronectin as described above. Following overnight treatment with 5μg/mL IgG or 9EG7, HUVECs were washed with PBS and fixed in 4% fresh paraformaldehyde for 10 minutes. Immunostaining was performed immediately following blocking with 10% horse serum in 1% BSA/PBS for 30 minutes at room temperature. Primary antibodies against VE-cadherin (R&D, AF938) and zonula occludens (ZO)-1 (ThermoFisher, 61–7300) were diluted in 1% bovine serum albumin (BSA)/PBS and added to the cells overnight at 4°C. Donkey-α-rabbit AlexaFluor 594 (A-21207) and donkey-α-goat AlexaFluor 647 (A-21447) secondary antibodies (both ThermoFisher) were used at a dilution of 1:300 in 1% BSA/PBS and cells were incubated for 1hr at room temperature. Cells were mounted using DAPI-containing mounting medium (Southern Biotech) and allowed to dry overnight before imaging using a Leica SP8 confocal microscope.
For analysis of junctional VE-cadherin and ZO-1 abundance, 5 representative images per treatment group for each of 3 experiments were analyzed. Using the ImageJ line tool, signal intensity along cell borders were measured at equally distributed areas, with a total of 25 measurements across each image, then averaged for comparison across treatment groups.

McCurdy S., Lin J., Shenkar R., Moore T., Lightle R., Faurobert E., Lopez-Ramirez M.A., Awad I, & Ginsberg M.H. (2022). β1 Integrin Monoclonal Antibody Treatment Ameliorates Cerebral Cavernous Malformations. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(12), e22629.

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VE-Cadherin Co-Immunoprecipitation from HUVECs

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HUVECs transfected with si-control or si-NRP1 and cultured under static conditions were lysed in 50 mM Tris, pH 8.0, 50 mM KCl, 1% (vol/vol) IGEPAL in the presence of protease inhibitor cocktail 2 and phosphatase inhibitor cocktail. 1.5 mg of protein was incubated with 3 μg goat anti-human VE-Cadherin (R&D #AF938) or control goat IgG (R&D # AB-108-C) and then with 30 μl magnetic protein G Dynabeads (Life Technologies) at 4°C overnight. Beads were collected with a Dynabead magnet, washed three times with lysis buffer on a rotating wheel at 4°C for 5 min, and resuspended in 50 μl Laemmli sample buffer for SDS-PAGE and immunoblotting.

Bosseboeuf E., Chikh A., Chaker A.B., Mitchell T.P., Vignaraja D., Rajendrakumar R., Khambata R.S., Nightingale T.D., Mason J.C., Randi A.M., Ahluwalia A, & Raimondi C. (2023). Neuropilin-1 interacts with VE-cadherin and TGFBR2 to stabilize adherens junctions and prevent activation of endothelium under flow. Science signaling, 16(786), eabo4863.

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Immunoblotting Protocols for Cellular Proteins

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Whole cell lysates for immunoblotting were prepared by dissolving cells in Laemmli sample buffer containing 5% of 2‐mercaptoethanol (Bio‐Rad). Primary antibodies used for immunoblotting include anti‐lamin A/C (MAB3211; Millipore, 1:500 dilution), progerin (Cao et al., 2011 (link)) with a dilution of 1:500, anti‐eNOS (D9A5L; Cell Signaling, 1:1000), anti‐pThr eNOS (Cell Signaling, 1:1000), mouse anti‐GAPDH (sc‐47724; Santa Cruz, 1:3000), goat anti‐VE‐cadherin (AF938; R&D Systems, 1:500), and anti‐PECAM‐1 (AF806, R&D Systems, 1:500), anti‐MMP‐1, MMP‐2, MMP‐7, MMP‐9, MMP‐14, TIMP1, and TIMP2 (Kit # 73959, Cell Signaling, 1:1000 dilution). Secondary antibodies include anti‐mouse (sc‐516102; Santa Cruz, 1:5000), anti‐rabbit (211–035–109; Jackson Immuno‐Research, 1:5000), anti‐goat (HAF017; R&D Systems, 1:1000), and anti‐goat (HAF019; R&D Systems, 1:1000 dilution).

Gete Y.G., Koblan L.W., Mao X., Trappio M., Mahadik B., Fisher J.P., Liu D.R, & Cao K. (2021). Mechanisms of angiogenic incompetence in Hutchinson–Gilford progeria via downregulation of endothelial NOS. Aging Cell, 20(7), e13388.

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Immunofluorescent Staining of EBs

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The EBs were fixed with 4% PFA in DPBS for 20 min for immunofluorescent staining, following our routine protocol.²⁶ (link) Primary antibodies included the following: Alexa Fluor 488 CD56 (1:200, MHCD5620, Thermo Fisher Scientific), VE-Cadherin (1:200, AF938, R&D Systems, Minneapolis, MN), and SOX17 (1:400, 698501, BioLegend, San Diego, CA, USA). Secondary antibodies included the following: Alexa Fluor 488 Donkey anti-Goat IgG (H+L) Secondary Antibody (1:200, A11055, Thermo Fisher Scientific) and Alexa Fluor 488 Goat anti-Mouse IgG (H+L) Secondary Antibody (1:200, A11029, Thermo Fisher Scientific).

Ruan J., Hirai H., Yang D., Ma L., Hou X., Jiang H., Wei H., Rajagopalan C., Mou H., Wang G., Zhang J., Li K., Chen Y.E., Sun F, & Xu J. (2019). Efficient Gene Editing at Major CFTR Mutation Loci. Molecular Therapy. Nucleic Acids, 16, 73-81.

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Cochlear Immunohistochemistry in Mice

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Cochleae were dissected from newborn (postnatal day 0, P0), P6, P12, P25 and P90 mice. After rapid decapitation, inner ears were removed and fixed for 2h in 4% formaldehyde. If necessary, tissues were decalcified prior to cryosectioning. Sections were cut parallel to the modiolus (mid-modiolar cut). Tissue sections of the cochlea were incubated overnight at 4 °C with primary antibodies directed against LDLR (rabbit monoclonal antibody [EP1553Y]; 1:50; Abcam; ab52818), myosin VIIa (mouse monoclonal antibody; 1:50; Santa Cruz Biotechnology; sc-74516), Kir4.1 (mouse monoclonal antibody; 1:50; Santa Cruz Biotechnology; sc-293252) and VE-cadherin (goat polyclonal antibody; 1:50; R&D Systems; AF938). The anti-LDLR antibody used in this study was found to give a positive signal in western-blot performed with liver extracts of wildtype mice, but not with liver extracts of Ldlr -/ -mice ( Li et al., 2021 ) (link). Tissues were then incubated for 1h with Rhodamine Red X-, Cy5-and/or FITC-conjugated anti-mouse, anti-goat and antirabbit IgGs secondary antibodies (Jackson Immunoresearch Laboratories). F-actin staining was obtained using Phalloidin-iFluor 647 reagent (1:50; Abcam; ab176759). Antibody specificity was tested by omitting each of the primary antibodies. Pictures were representative of three age-matched cochleae, from three different animals.

Saume A., Thiry M, & Defourny J. (2021). LDLR expression in the cochlea suggests a role in endolymph homeostasis and cochlear amplification. Hearing research, 409.

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