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Ab15348

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Ab15348 is an Abcam product that functions as a laboratory equipment. It is designed for use in scientific research applications.

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Lab products found in correlation

Ab92323, by Abcam (9 mentions) Hoechst 33342, by Thermo Fisher Scientific (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Pvdf membrane, by Bio-Rad (3 mentions) Ab150075, by Abcam (3 mentions) Ab28364, by Abcam (3 mentions) Ab8245, by Abcam (3 mentions) Ab49900, by Abcam (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Fluoroshield, by Merck Group (2 mentions) Alexa fluor 536 anti rabbit, by Thermo Fisher Scientific (2 mentions) Fluormount, by Merck Group (2 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions) Ab8227, by Abcam (2 mentions) Mabf2158, by Merck Group (2 mentions) Ab228023, by Abcam (2 mentions) Ab104139, by Abcam (2 mentions) Triton x 100, by Merck Group (2 mentions) Epr3861, by Abcam (2 mentions) Triton x 100 sample buffer, by Merck Group (2 mentions)

Close spelling variants of this product

ACE2 ab15348 (2 citations)

72 protocols using ab15348

1

Western Blot Analysis of ACE-2 and SARS-CoV-2 Spike

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Total protein lysate was analyzed by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad). Anti-ACE-2 antibody (1:1000, ab15348, Abcam, Cambridge, UK) and/or anti-SARS-CoV-2 (2019-nCoV) spike (S) antibody (1:1000, 40591-MM42, Sino Biological, Wayne, NJ, USA) were probed with the appropriate secondary antibodies (Cell Signaling, Danvers, NJ, USA) and loading control (anti-β-actin antibody; 1:2000, 4970, Cell Signaling). Chemiluminescence signals were acquired by ImageQuantTM LAS 4000 (GE Healthcare Europe GmbH, Milan, Italy).
Lotti V., Merigo F., Lagni A., Di Clemente A., Ligozzi M., Bernardi P., Rossini G., Concia E., Plebani R., Romano M., Sbarbati A., Sorio C, & Gibellini D. (2022). CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells. Cells, 11(8), 1347.
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2

Immunofluorescence Analysis of SARS-CoV-2 Proteins

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Ten-micrometer-thick cryosections were double-stained using a monoclonal mouse anti-SARS-CoV-2 antibody (1:1000 dilution, #MBS569903; MyBioSource, San Diego, CA, USA) and one of the following antibodies: polyclonal rabbit anti-pan cytokeratin antibody (1:100 dilution, #ab9377; Abcam), monoclonal rabbit anti-CD14 antibody (1:100 dilution, #ab18332; Abcam), polyclonal rabbit anti-ACE2 (1:100 dilution, recognizing both short and long forms of ACE2; #PK-AB718-3217, PromoCell, Heidelberg, Germany), monoclonal rabbit anti-ICAM-1 (1:100 dilution, #ab109361, Abcam). Mouse monoclonal and rabbit monoclonal or polyclonal isotype antibodies (#ab18469, #ab172730 and #ab15348; Abcam) functioned as negative controls. In the secondary step, FITC-conjugated polyclonal goat anti-mouse antibodies (1:200 dilution #F2761, ThermoFisher Scientific) were combined with Texas Red-conjugated polyclonal donkey anti-rabbit antibodies (1:100 dilution, #ab6800, Abcam). In the tertiary step, FITC-conjugated polyclonal donkey anti-goat antibodies were added (1:200 dilution, #A16006, ThermoFisher Scientific). DAPI (#D9542, Sigma-Aldrich) was used to counterstain cell nuclei. Slides were mounted with Fluoroshield™ (#F6182, Sigma-Aldrich) and analyzed using a Leica (TCS SPE) confocal microscope and Leica Las X v3.7.2.22383 software.
Van Cleemput J., van Snippenberg W., Lambrechts L., Dendooven A., D’Onofrio V., Couck L., Trypsteen W., Vanrusselt J., Theuns S., Vereecke N., van den Bosch T.P., Lammens M., Driessen A., Achten R., Bracke K.R., Van den Broeck W., Von der Thüsen J., Nauwynck H., Van Dorpe J., Gerlo S., Maes P., Cox J, & Vandekerckhove L. (2021). Organ-specific genome diversity of replication-competent SARS-CoV-2. Nature Communications, 12, 6612.
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3

ACE2-SARS-CoV-2 Spike Protein Interaction Assay

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Cultured cells were harvested and lysed in 1% Triton X-100 in PBS. Equivalent protein quantities were immunoprecipitated with anti-ACE2 antibody (Abcam, ab15348)-conjugated magnetic beads (Dynabeads™ Protein A; Thermo Fisher Scientific, 10002D) for 90 minutes at RT. Immunoprecipitants were eluted and subjected to immunoblotting with anti-ACE2 antibody (1:1000, Cell Signaling, #15983) and anti-SARS-CoV-2 Spike protein antibody (1:2000, Abcam, ab275759).
Liu B., Liao J., Rao X., Kushner S.A., Chung C.D., Chang D.D, & Shuai K. (1998). Inhibition of Stat1-mediated gene activation by PIAS1. Proceedings of the National Academy of Sciences of the United States of America, 95(18), 10626-10631.
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4

Visualizing ACE2 and Smooth Muscle Actin in Tissue

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For immunofluorescence staining, sections underwent deparaffination and subsequently a heat-mediated antigen retrieval in sodium-citrate buffer (10 mM sodium-citrate, 0.05% Tween 20, pH 6,0). Upon washing with PBS and incubation with blocking buffer (3% BSA, 0.1% TritonX, 0.05% Tween 20), slides were incubated with primary antibodies anti-ACE2 (ab15348, Abcam) and anti-alpha smooth muscle actin (ab5694, Abcam) overnight. AlexaFluor488 (A11001, Invitrogen) and AlexaFluor568 (A11011, Invitrogen) were used as secondary antibodies. For counterstaining, DAPI was used (62248, Thermo Scientific). Sections were z-stack imaged with a LSM980 confocal microscope (Carl Zeiss, Germany). Subsequent 3D rendering was performed using the Imaris software Version 9.6 (Bitplane AG, Switzerland). Images were analyzed automatedly as described previously 16 (link).
Nägele F., Graber M., Hirsch J., Pölzl L., Sahanic S., Fiegl M., Hau D., Engler C., Lechner S., Stalder A.K., Mertz K.D., Haslbauer J.D., Tzankov A., Grimm M., Tancevski I., Holfeld J, & Gollmann-Tepeköylü C. (2022). Correlation between structural heart disease and cardiac SARS-CoV-2 manifestations. Communications Medicine, 2, 142.
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5

Protein Expression Analysis in Endothelial Cells

Cited 1 time
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After treatment, BAECs were lysed in lysis buffer (20 mmol/l Tris–HCl pH 7.4, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l sodium orthovanadate, and 1% Triton X-100, supplemented with protease inhibitor cocktail) for 20 min on ice and then supernatant separated by centrifugation at 12,000 rpm for 10 min at 4°C. Following protein concentration determination by DC protein assay (#5000112, Bio-Rad, Hercules, CA, United States), 25–40 μg of protein were separated in 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by standard Western blotting protocol by probing with antibodies for ACE2 (1:1,000, #ab15348, Abcam, Waltham, MA, United States), TMPRSS2 (1:1,000, #ab92323, Abcam, Waltham, MA, United States), NOX2 (1:250, #611414, BD Biosciences, San Jose, CA, United States), MCP-1 (1:500, #ab9669, Abcam, Waltham, MA, United States), and β-actin (1:1,000, #A2066, MilliporeSigma, St. Louis, MO, United States) as we previously published (23 (link)). The protein bands were visualized by enhanced chemiluminescent methods, and band densities quantified using National Institutes of Health (NIH) Image J program.
Youn J.Y., Wang J., Li Q., Huang K, & Cai H. (2022). Robust therapeutic effects on COVID-19 of novel small molecules: Alleviation of SARS-CoV-2 S protein induction of ACE2/TMPRSS2, NOX2/ROS, and MCP-1. Frontiers in Cardiovascular Medicine, 9, 957340.
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6

Measuring ACE2 Expression in Cells

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Cells were seeded on glass coverslips precoated with collagen in 24-well plates. After incubation at 37°C, cells were treated according to the experimental protocol with E2 (200 nM), raloxifene (20μM), and S (10 ng/ml). After 72 hours, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained overnight at 4°C with ACE2 protein–specific antibody (Abcam, Ab15348). Cells were then incubated with anti-rabbit secondary antibody (Alexa Fluor 536 anti-rabbit, Invitrogen Life Technologies) for 1 hour at 37°C. Nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific) for nuclear staining for 20 min. Cells were mounted with Fluor mount (Sigma-Aldrich, St. Louis, MO, USA), and images were acquired through confocal microscope LSM 800, 60× magnification, software ZEN 2.1 blue edition (Carl Zeiss, Jenza, Germany) and analyzed with ImageJ software.
Solis O., Beccari A.R., Iaconis D., Talarico C., Ruiz-Bedoya C.A., Nwachukwu J.C., Cimini A., Castelli V., Bertini R., Montopoli M., Cocetta V., Borocci S., Prandi I.G., Flavahan K., Bahr M., Napiorkowski A., Chillemi G., Ooka M., Yang X., Zhang S., Xia M., Zheng W., Bonaventura J., Pomper M.G., Hooper J.E., Morales M., Rosenberg A.Z., Nettles K.W., Jain S.K., Allegretti M, & Michaelides M. (2022). The SARS-CoV-2 spike protein binds and modulates estrogen receptors. Science Advances, 8(48), eadd4150.
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7

ACE2 Protein Expression Analysis

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MLE-12 cells were transfected with 2 μg of pCMV-ACE2-GFPSpark plasmid or control pCMV3 plasmid per 106 cells using TransIT-2020 Transfection Reagent (Mirus, USA). Protein extracts were prepared at 48 h post-transduction using RIPA lysis buffer. Lung tissue samples were harvested and mechanically homogenized using a TGrinder instrument (Tiangen Biotechnologies, China). Protein extracts were prepared using a tissue protein extraction kit (BC3790; Solarbio, China). Equal amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were stained with rabbit anti-human ACE2 polyclonal antibody (ab15348; Abcam, UK) or rabbit anti-β-actin polyclonal antibody (ab8227; Abcam, UK). Proteins were detected using the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, USA).
Liang Y., Li H., Li J., Yang Z.N., Li J.L., Zheng H.W., Chen Y.L., Shi H.J., Guo L, & Liu L.D. (2020). Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model. Zoological Research, 41(6), 621-631.
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8

Immunohistochemistry of ACE2, EPCAM and TMPRSS2

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Specimens were obtained via clinical endoscopy during routine care (Table S1, S2). Tissue was formalin fixed and paraffin embedded by the clinical pathology core at our institution. Primary antibodies used included ACE2 (abcam-ab15348, 1:1000), EPCAM (abcam-ab228023, prediluted) and mouse anti-TMPRSS2 (Millipore-MABF2158, 1:500) and staining was performed as detailed in supplementary methods.
Suárez-Fariñas M., Tokuyama M., Wei G., Huang R., Livanos A., Jha D., Levescot A., Irizar H., Kosoy R., Cording S., Wang W., Losic B., Ungaro R.C., Di’Narzo A., Martinez-Delgado G., Suprun M., Corley M.J., Stojmirovic A., Houten S.M., Peters L., Curran M., Brodmerkel C., Perrigoue J., Friedman J.R., Hao K., Schadt E.E., Zhu J., Ko H.M., Cho J., Dubinsky M.C., Sands B.E., Ndhlovu L., Cerf-Bensusan N., Kasarskis A., Colombel J.F., Harpaz N., Argmann C, & Mehandru S. (2020). Intestinal Inflammation Modulates the Expression of ACE2 and TMPRSS2 and Potentially Overlaps With the Pathogenesis of SARS-CoV-2–related Disease. Gastroenterology, 160(1), 287-301.e20.
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9

Visualizing SARS-CoV-2 Receptor Expression

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ACE2 knockout and DPP4 knockout Calu-3 cells (2.0 × 105/well) were plated separately onto cover glasses in a 9 cm2 well of the plates, grown overnight (to ∼5 × 105 cells), and transfected with Wt-RBD-Fc-IgG1 (Addgene # 141183) construct using lipofectamine 3000 (Invitrogen) following manufacturer's instructions. 0.8 μg plasmid was used for transfection per 5 × 105 cells in each 4 cm2 well of the plate. After 8 h post-transfection, cells were fixed in formaldehyde for 5 min and permeabilized with PBS containing 0.1 % Triton X-100. Fixed cells were blocked in PBS containing 1 % BSA for 1 h. Afterward, cells were incubated with either a primary antibody specific to DPP4 or ACE2 (Abcam, ab15348) or an anti-IgG1-Fc-AF488-labelled antibody (Invitrogen, A10631), followed by an alexa-fluor 647 tagged anti-rabbit secondary antibody (Abcam, ab150079) specific to primary antibody of DPP4 or ACE2. Mounting was done using fluoroshield (Sigma), and cells were visualized under a confocal laser scanning inverted microscope, Carl-Zeiss LSM980 (63×)2.
Mani S., Kaur A., Jakhar K., Kumari G., Sonar S., Kumar A., Das S., Kumar S., Kumar V., Kundu R., Pandey A.K., Singh U.P, & Majumdar T. (2023). Targeting DPP4-RBD interactions by sitagliptin and linagliptin delivers a potential host-directed therapy against pan-SARS-CoV-2 infections. International Journal of Biological Macromolecules, 245, 125444.
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10

Immunofluorescence and Immunoblot Analysis of COVID-19 Related Proteins

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Immunofluorescence analysis was performed for actin (AlexaFluor 488 Phalloidin, #A12379, Thermo Fisher Scientific, Waltham, MA, United States), GFP (#ab6556, Abcam, Cambridge, United Kingdom) V5 tag (#37-7,500, Thermo Fisher Scientific, Waltham, MA, United States), TMEM16F (ab256302, Abcam, Cambridge, United Kingdom), and ACE2 (ab87436 and ab15348, both from Abcam, Cambridge, United Kingdom). Fluorescent secondary antibodies were obtained from Sigma-Aldrich, Burlington, MA, United States. Immunohistochemistry for platelets was performed with antibody 760–4,249 (Roche, Basel, Switzerland) against CD61.
Immunoblots were performed with primary antibodies against Spike (#GTX632604, Genetex, San Antonio, TX, United States), TMEM16F (#HPA038958, Sigma-Aldrich, Burlington, MA, United States) and tubulin (Cell Signaling, #3873S). Anti-rabbit and anti-mouse HRP-conjugated antibody were obtained from Abcam, Cambridge, United Kingdom.
Cappelletto A., Allan H.E., Crescente M., Schneider E., Bussani R., Ali H., Secco I., Vodret S., Simeone R., Mascaretti L., Zacchigna S., Warner T.D, & Giacca M. (2023). SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity. Frontiers in Cardiovascular Medicine, 9, 1013262.
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