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Ab181153

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Ab181153 is a laboratory equipment product. It is a device used for the analysis or manipulation of samples in a laboratory setting. The core function of this product is to perform specific laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

Ab40873, by Abcam (2 mentions) Ab106926, by Abcam (2 mentions) Ab90716, by Abcam (2 mentions) Alexa fluor 488 igg 53 5381 80, by Thermo Fisher Scientific (2 mentions) Igg h l rhodamine red conjugate r 6394, by Thermo Fisher Scientific (2 mentions) Hoechst, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions) Ab65080, by Abcam (2 mentions) Ab69090, by Abcam (2 mentions) Ab23707, by Abcam (1 mentions) Ab56783, by Abcam (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Clarity ecl substrate, by Bio-Rad (1 mentions) Criterion tgx gel, by Bio-Rad (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Trans blot turbo system, by Bio-Rad (1 mentions) Pa5 27137, by Thermo Fisher Scientific (1 mentions) Ab22048, by Abcam (1 mentions) Anti citrate synthase, by Cell Signaling Technology (1 mentions)

8 protocols using ab181153

1

Immunofluorescence Staining of Lung Cells

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Briefly, tissues were stained with goat polyclonal IgG to Irp2 (ab106926, Abcam) at a 1:50 dilution or rabbit monoclonal IgG to Irp2 (ab181153, Abcam) at a 1:50 dilution. Co-staining for type II epithelial cells was carried out using a rabbit polyclonal IgG to prosurfactant protein C (Pro-SPC) (ab90716, Abcam) at a 1:50 dilution and for type I epithelial cells using anti-mouse podoplanin alexa Fluor® 488 IgG (53-5381-80, Affymetrix) at a 1:500 dilution. Co-staining for Irp2 and markers of ciliated airway epithelial cells was conducted using a rabbit IgG for acetyl-α-tubulin (Lys40) (D20G3, Cell Signaling) at a 1:50 dilution, and co staining for non-ciliated secretory epithelial cells was conducted using a rabbit polyclonal IgG to uteroglobin (ab40873, Abcam) at a 1:100 dilution. Secondary staining was carried out using goat anti-rabbit IgG (H+L) rhodamine red conjugate (R-6394, Life Technologies) at a 1:500 dilution or donkey anti-goat IgG (H+L) secondary antibody, alexa Fluor® 488 conjugate (A-11055, Life technologies) at a 1:500 dilution. Nuclei were counter-stained using TO-PRO®-3 Iodide (T3605, Life Technologies) at a 1:1000 dilution or Hoechst (33342, ThermoFisher Scientific) at a 1:300 dilution.
Cloonan S.M., Glass K., Laucho-Contreras M.E., Bhashyam A.R., Cervo M., Pabón M.A., Konrad C., Polverino F., Siempos I.I., Perez E., Mizumura K., Ghosh M.C., Parameswaran H., Williams N.C., Rooney K.T., Chen Z.H., Goldklang M.P., Yuan G.C., Moore S.C., Demeo D.L., Rouault T.A., D’Armiento J.M., Schon E.A., Manfredi G., Quackenbush J., Mahmood A., Silverman E.K., Owen C.A, & Choi A.M. (2016). Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice. Nature medicine, 22(2), 163-174.
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2

Mitochondrial Stress Response Evaluation

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MSCs exposed to normoxia (5%) or hyperoxia (85%) for 24 h were lysed in RIPA buffer (Alfa Aesar. Cat. No. J62885) and the lysate was centrifuged at 6,000 g for 10 min at 4ºC, then the supernatant was mixed with 4 × Laemmli sample buffer for western blotting according to standard procedures. 30 to 60 µg of total protein from the samples were separated on 4–20% Criterion TGX Gels (Bio-Rad, Cat. No. 5678093), transferred to PVDF membranes (Trans-Blot Turbo System, Bio-Rad) and probed with antibodies against PINK1 (ab23707), Aconitase (ab181153),TOM20 (ab56783, Abcam, MA) and B-Actin (Cell Signaling Technology, 5125S) as loading control61 (link). Membranes were developed using Clarity ECL Substrate (Bio-Rad) and visualized with ChemiDoc imaging System. Densitometry analysis of band intensities was performed using ImageJ software.
Hazra S., Li R., Vamesu B.M., Jilling T., Ballinger S.W., Ambalavanan N, & Kandasamy J. (2022). Mesenchymal stem cell bioenergetics and apoptosis are associated with risk for bronchopulmonary dysplasia in extremely low birth weight infants. Scientific Reports, 12, 17484.
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3

Ferroptosis Marker Analysis in BMVEC Lysates

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Cell lysates of BMVECs was assessed for ferroptosis markers. Briefly, equivalent amounts of cell lysates of BMVECs (15 μg protein/lane) were loaded onto 10% SDS-PAGE, proteins separated, and proteins transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin followed by incubation for overnight at 4°C with primary antibody anti-Ferritin heavy chain (ab65080, Abcam), anti-ferritin light chain (ab69090, Abcam), anti-ACSL4 (Acyl-CoA synthetase long-chain family member 4) (PA5–27137, Invitrogen), anti-IREB2 (iron responsive element binding protein 2) (ab181153, Abcam) at 1:1000 dilution or anti β-actin (A2854,Sigma) at 1:3000 dilution. After washing, membranes were incubated for 1 h at 20°C with appropriate secondary antibodies (horseradish peroxidase [HRP]-conjugated; dilution 1:3000). Pre-stained molecular weight markers were run in parallel to identify the molecular weight of proteins of interest. For chemiluminescent detection, the membranes were treated with enhanced chemiluminescent reagent and the signals were monitored on FluorChem-E Imager (Protein Simple, San Jose, CA). Relative band intensity was determined by densitometry software and normalized with β-actin protein.
Abdul Y., Li W., Ward R., Abdelsaid M., Hafez S., Dong G., Jamil S., Wolf V., Johnson M.H., Fagan S.C, & Ergul A. (2020). Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis. Translational stroke research, 12(4), 615-630.
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4

Immunofluorescence Staining of Brain Sections

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Paraformaldehyde fixed frozen brain sections or BMVECs grown on slides were subsequently washed with TBS followed by treatment with 0.2% Triton X-100 for 3 minutes. After washing, samples were blocked by 5% BSA for 1 hour at room temperature. Slides were then incubated with primary antibodies like anti-IREB2 (iron responsive element binding protein 2) (ab181153, Abcam), anti-4 hydroxynonenal (anti-4HNE) (ab46545, Abcam), anti-TLR4 (toll like receptor 4) (ab22048, Abcam) and anti-citrate synthase (14309, Cell Signaling) at a 1:100 dilution in 0.2% BSA at 4°C overnight. Cells were washed and incubated with AlexaFlour 488 conjugated secondary antibody (Jackson Immuno Research Laboratories, Inc., West Grove, PA) at a 1:400 dilution at room temperature for 1 hour. Negative control slides were incubated with 0.2% BSA in place of the primary antibody. Slides were imaged on Axiovert 200 microscope (Carl Zeiss MicroImaging, Thornwood, NY).
Abdul Y., Li W., Ward R., Abdelsaid M., Hafez S., Dong G., Jamil S., Wolf V., Johnson M.H., Fagan S.C, & Ergul A. (2020). Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis. Translational stroke research, 12(4), 615-630.
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5

Protein Expression Profiling in Duodenum, Liver, and Serum

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Duodenal epithelial scrapings, liver (ground by mortar and pestle), and serum samples (vehicle and 30 μg/kg TCDD groups) were sonicated on ice in RIPA buffer supplemented with protease inhibitor cocktail (Sigma). Samples were centrifuged and total protein in the supernatant was measured using the bicinchoninic acid (BCA) assay (Sigma). The WES capillary electrophoresis system (ProteinSimple, San Jose, CA) was used with the following antibodies: ACO1 (1:65; ab126595; Abcam, Cambridge, MA), FTH1 (1:65; ab65080; Abcam), FTL1 (1:65; ab69090; Abcam), HAMP/HAMP2 (1:10; ab190775; Abcam), HAO1 (1:30; ab93137; Abcam), HP (1:650; ab131236; Abcam), HPX (1:10; ab90947; Abcam), IREB2 (1:65; ab181153; Abcam), SLC11A2 (DMT1; 1:10; ab123085; Abcam), SLC40A1 (1:30; NBP1-21502; Novus Biologicals, Littleton, CO), TFRC (1:30; ab84036; Abcam), and TRF (1:30; ab82411; Abcam). Primary antibodies were detected using a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Chemiluminescence signals were analyzed with Compass software (ProteinSimple). Protein levels in the duodenum and liver were normalized to SDHA levels, while protein levels in the serum were normalized to sample volume.
Fader K.A., Nault R., Kirby M.P., Markous G., Matthews J, & Zacharewski T.R. (2017). Convergence of Hepcidin Deficiency, Systemic Iron Overloading, Heme Accumulation, and REV-ERBα/β Activation in Aryl Hydrocarbon Receptor-Elicited Hepatotoxicity. Toxicology and applied pharmacology, 321, 1-17.
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6

Antibody Panel for Peroxisomal Protein Detection

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Rabbit FLAG antibody (#14793, Cell Signaling Technology), mouse CAT antibody (YIF-LF-MA0003, Biomol International), mouse GAPDH antibody (AM4300, Invitrogen), rabbit PGD antibody (#13389, Cell Signaling Technology), rabbit TKT antibody (#8616, Cell Signaling Technology), rabbit NRF2 antibody (#12721, Cell Signaling Technology), rabbit CAT antibody (ab16731, Abcam), rabbit PMP70 antibody (ab3421, Abcam), rabbit PEX12 antibody (ab103456, Abcam), rabbit PGLS antibody (ab127560, Abcam), rabbit β-actin antibody (ab8227, Abcam), rabbit IREB2 antibody (ab181153, Abcam), mouse PMP70 antibodies (SAB4200181, Sigma). Rabbit antibodies to PEX5 (Otera et al., 2000 (link)), PEX13 (Mukai and Fujiki, 2006 (link)), PEX14 (Shimizu et al., 1999 ), ACOX1 (Tsukamoto et al., 1990 (link)), LONP2 (Okumoto et al., 2011 ) were used as described in corresponding references.
Dubreuil M.M., Morgens D.W., Okumoto K., Honsho M., Contrepois K., Lee-McMullen B., Traber G.M., Sood R.S., Dixon S.J., Snyder M.P., Fujiki Y, & Bassik M.C. (2020). Systematic Identification of Regulators of Oxidative Stress Reveals Non-canonical Roles for Peroxisomal Import and the Pentose Phosphate Pathway. Cell reports, 30(5), 1417-1433.e7.
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7

Immunoblotting Analysis of Iron Regulatory Proteins

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Samples were lysed in RIPA-buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycolic acid) containing protease inhibitor (Complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics) and centrifuged at 12,300 rpm for 10 min at 4 °C and supernatants were collected. Total protein concentration was determined by BCA protein assay (Pierce). Aliquots of homogenate with equal protein concentrations were separated in 10% acrylamide gel and transferred to nitrocellulose membranes by mini trans-blot (Bio-Rad). The membranes were blocked with milk (5% w/v) and probed with appropriate primary and secondary IgG-HRP conjugated antibodies (Millipore). Enhanced chemiluminescence detection system (GE Healthcare) was used for developing on autoradiography-films (GE Healthcare). Densitometric quantification was performed using ImageJ and normalized to the relative amount of α-Tubulin and expressed as n-fold of control samples. In this study the following antibodies were used: Anti-IRP2 (1:1000, Abcam, ab181153), Anti-TFRC (1:500, Abcam Cat# ab84036 RRID:AB_10673794), Anti-FBXL5 (1:500, Abcam Cat# ab68069 RRID:AB_1140312), α-TUB (1:20000, Sigma-Aldrich Cat# T5168 RRID:AB_477579); secondary HRP antibodies: goat-anti rabbit (1:2000, Santa Cruz Biotechnology Cat# sc-2004 RRID:AB_631746), goat-anti mouse (1:2000, Santa Cruz Biotechnology Cat# sc-2005 RRID:AB_631736).
Ripa R., Dolfi L., Terrigno M., Pandolfini L., Savino A., Arcucci V., Groth M., Terzibasi Tozzini E., Baumgart M, & Cellerino A. (2017). MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging. BMC Biology, 15, 9.
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8

Immunofluorescence Staining of Lung Cells

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Briefly, tissues were stained with goat polyclonal IgG to Irp2 (ab106926, Abcam) at a 1:50 dilution or rabbit monoclonal IgG to Irp2 (ab181153, Abcam) at a 1:50 dilution. Co-staining for type II epithelial cells was carried out using a rabbit polyclonal IgG to prosurfactant protein C (Pro-SPC) (ab90716, Abcam) at a 1:50 dilution and for type I epithelial cells using anti-mouse podoplanin alexa Fluor® 488 IgG (53-5381-80, Affymetrix) at a 1:500 dilution. Co-staining for Irp2 and markers of ciliated airway epithelial cells was conducted using a rabbit IgG for acetyl-α-tubulin (Lys40) (D20G3, Cell Signaling) at a 1:50 dilution, and co staining for non-ciliated secretory epithelial cells was conducted using a rabbit polyclonal IgG to uteroglobin (ab40873, Abcam) at a 1:100 dilution. Secondary staining was carried out using goat anti-rabbit IgG (H+L) rhodamine red conjugate (R-6394, Life Technologies) at a 1:500 dilution or donkey anti-goat IgG (H+L) secondary antibody, alexa Fluor® 488 conjugate (A-11055, Life technologies) at a 1:500 dilution. Nuclei were counter-stained using TO-PRO®-3 Iodide (T3605, Life Technologies) at a 1:1000 dilution or Hoechst (33342, ThermoFisher Scientific) at a 1:300 dilution.
Cloonan S.M., Glass K., Laucho-Contreras M.E., Bhashyam A.R., Cervo M., Pabón M.A., Konrad C., Polverino F., Siempos I.I., Perez E., Mizumura K., Ghosh M.C., Parameswaran H., Williams N.C., Rooney K.T., Chen Z.H., Goldklang M.P., Yuan G.C., Moore S.C., Demeo D.L., Rouault T.A., D’Armiento J.M., Schon E.A., Manfredi G., Quackenbush J., Mahmood A., Silverman E.K., Owen C.A, & Choi A.M. (2016). Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice. Nature medicine, 22(2), 163-174.
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