Equal amounts of protein sample were prepared in SDS-sample buffer and heated for 10 minutes at 100°C. Cleared lysates were electrophoresed in 10% polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked in TBST with 5% skim milk powder to prevent non-specific binding. Membranes were probed with rabbit polyclonal anti-UCP1 1:1000 (#ab23841, Abcam, Cambridge, UK), and anti-rabbit IgG HRP-linked antibody 1:5000 (#7074, Cell Signalling Technology, Qld, Australia). Density of protein signal was normalized to that of the internal control, 14-3-3 1:2000 (Sc-629 Santa Cruz, CA, USA). Protein expression was visualized using enhanced chemiluminescence, and densitometry of UCP1 and 14-3-3 bands was performed using ImageJ software (NIH, Bethesda, MD, USA).
Ab23841
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab23841 is a mouse monoclonal antibody that targets a specific protein. This antibody is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Hematoxylin and eosin, by Merck Group (2 mentions)
Sigmafast dab with metal enhancer, by Merck Group (2 mentions)
Zen 2012, by Zeiss (2 mentions)
Ab6640, by Abcam (2 mentions)
Ab10983, by Abcam (2 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ab126611, by Abcam (1 mentions)
Ab32358, by Abcam (1 mentions)
Anti lc3b l7543, by Merck Group (1 mentions)
Rapamycin r8781, by Merck Group (1 mentions)
Monoclonal antibody to β actin, by Merck Group (1 mentions)
Comprehensive lab animal monitoring system, by Columbus Instruments (1 mentions)
Alexa fluor 680 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti ucp1 antibody, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Phosphatase inhibitor mixture, by Merck Group (1 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Dnmt3b, by Santa Cruz Biotechnology (1 mentions)
Mitochondrial total oxphos protein antibody set, by Abcam (1 mentions)
36 protocols using ab23841
1
Western Blot Analysis of UCP1 Protein
2
Adipose Tissue Histology Analysis
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Brown adipose tissue and inguinal and epididymal adipose tissues were fixed in 4% formaldehyde overnight at room temperature, embedded in paraffin, and cut into 5-μm section with a microtome. Slides were deparaffinized, rehydrated, and stained with hematoxylin and eosin (Sigma) using a standard protocol. Alternatively, sections were stained with anti-UCP1 (ab23841, Abcam; 1:200) and developed with SIGMAFAST DAB with Metal Enhancer (Sigma). Sections were examined by light microscopy (Motic BA600) and photographed with Moticam Pro 285A. Photomicrographs were scanned with an Abaton Scan 300/Color scanner.
3
Adiponectin Signaling and Autophagy Regulation
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Antibodies against phospho-AMPK (2531) at Thr172, AMPK (2603), phospho-IKKα (Ser176)/IKKβ (Ser177; 2078S), IKKα (2682), IKKβ (2370), phospho-IκB at Ser32/36 (9246), IκB (4814), phospho-ULK1 at Ser317 (6887), and ULK1 (8054) were from Cell Signaling. Anti-PGC1α (ST1204) was purchased from Millipore. Antibodies against AdipoR1 (ab126611), AdipoR2 (ab77612), UCP1 (ab23841), and C/EBPβ (ab32358) were from Abcam. Anti-LC3B (L7543) from Sigma was used for Western blot, analysis and Anti-LC3 pAb (PM036) from MBL International was used for staining. Anti-adiponectin and anti-β-tubulin were kindly provided by Dr. Lily Dong (University of Texas Health Science Center at San Antonio, San Antonio, TX) as described previously (Alsted et al., 2009 (link); Meng et al., 2017 (link)). AdipoRon (924416–43-3) and 5Z (253863–19-3) were obtained from Cayman Chemical Company. IRAK 1/4 inhibitor I (I5409) and rapamycin (R8781) were from Sigma-Aldrich. 3-MA (189490) was purchased from EMD Millipore. Two recombinant mouse adiponectin (full-length and mutated C39A) proteins (ab62957 and ab94676) were purchased from Abcam. Mouse ST2/IL-1 R4 antibody (MAB10041) was from R&D Systems.
4
Antibodies and Reagents for BMP Signaling
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Monoclonal antibodies against phospho-SMAD1/5/8 (#9511, Lot#14, 1:1,000), SMAD3 (#C25A9, Lot#13, 1:1,000) and BMPR2 (#6979, Lot#1, 1:1,000) were obtained from Cell Signaling. Monoclonal antibodies against BMPR1A (MAB2406, Lot#UEJ0112071, 1 μg ml−1) and BMPR1B (MAB505, Lot#DGD0410091, 1 μg ml−1) were obtained from R&D Systems. Monoclonal Antibody to β-Actin was obtained from Sigma Aldrich (#A2228, Lot#052M4816V, 1:1,000). Rabbit polyclonal antibody against UCP1 was obtained from Abcam (ab23841, Lot#GR34748-1, 5 μg ml−1). Recombinant human BMP7 (354-BP, Lot#EOS1914031, 3 nM l−1) was obtained from R&D Systems. Soluble form of LR11 protein (sLR11) was purified from the medium incubated with Colo201 (B226, JCRB Cell Bank, Osaka, Japan) by affinity column using monoclonal antibody against sLR11, followed gel filtration as previously described20 (link). Purified sLR11 was concentrated and used for experiments at a concentration of 10 ng ml−1, estimated from its biological activity in stimulating the migration of smooth muscle cells as previously described11 (link).
5
Immunohistochemical Analysis of UCP1 in BAT and WAT
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Tissues were harvested, fixed in 4% paraformaldehyde (pH 7.4) for 24 h at 4 °C and subsequently embedded in paraffin. Immunohistochemical analysis was performed on BAT and WAT sections rabbit anti-UCP1 polyclonal antibody (ab23841; Abcam, Cambridge, UK). Sections were rinsed thoroughly and incubated with labelled polymer HRP anti-rabbit (Dako Envision™+; Dako, Hamburg, Germany) for 1 h. Visualization was performed with 3,3′-diaminobenzidine. Microscopic examination was performed using an Axio Observer Microscop (Carl Zeiss, Jena, Germany). Images were obtained using ZEN2012 software (Carl Zeiss, Germany).
6
Adipose Tissue Histology and Calorimetry
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Brown adipose tissue and inguinal and epididymal adipose tissues were fixed in 4% formaldehyde overnight at room temperature, embedded in paraffin, and sectioned (5 μm) by microtome. The slides were deparaffinized, rehydrated, and stained with hematoxylin and eosin (Sigma) by a standard protocol. Alternatively, the sections were stained with anti-UCP1 (ab23841, Abcam; 1:200) and developed with SIGMAFAST DAB with Metal Enhancer (Sigma). Sections were examined by light microscopy (Motic BA600) and photographed with Moticam Pro 285A. Photomicrographs were scanned with an Abaton Scan 300/Color scanner.
Indirect Calorimetry and Calculated Energy Expenditure Whole-body oxygen consumption was measured with an open-circuit indirect calorimetry system with automatic temperature and light controls (Comprehensive Lab Animal Monitoring System, Columbus Instruments). Mice had ad libitum access to chow and water in respiration chambers, and data were recorded for 48 h, including 24 h of acclimatization. Energy expenditure was calculated as recommended by the manufacturer.
Indirect Calorimetry and Calculated Energy Expenditure Whole-body oxygen consumption was measured with an open-circuit indirect calorimetry system with automatic temperature and light controls (Comprehensive Lab Animal Monitoring System, Columbus Instruments). Mice had ad libitum access to chow and water in respiration chambers, and data were recorded for 48 h, including 24 h of acclimatization. Energy expenditure was calculated as recommended by the manufacturer.
7
Immunoblotting for Protein Detection in Fat Tissues
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Immunoblotting for protein detection was conducted as we described [27 (link),32 (link)]. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Tissue lysates were resolved by SDS-PAGE gels. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies used were as follows: Anti-UCP1 antibody (1:500, ab23841, Abcam, Cambridge, MA, USA); Anti-α-Tubulin antibody (1:1000, ABCENT4777, Advanced BioChemicals, Lawrenceville, GA, USA); Mitochondrial total OXPHOS protein antibody set (Abcam,ab110413); and Anti-pHSL (4126s, Cell Signaling Technology, Danvers, MA, USA); DNMT3b (sc-393845, Santa Cruz, Dallas, TX, USA, sc-393845); HSL (Cell Signaling, 4107s).
8
Immunohistochemical Analysis of UCP1 in Adipose Tissue
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IngWATs were fixed in 10% formalin and paraffin embedded. Multiple 5 μm sections were prepared and stained for UCP1. Briefly, sections were deparaffinized and rehydrated, followed by an antigen retrieval step in a modified citrate buffer (Dako Target Retrieval Solution, pH 6.1, Agilent). To reduce autofluorescence signal in adipose tissue, sections were then incubated in Sudan Black (0.3% in 70% ethanol). This was followed by blocking in Millipore blocking reagent (EMD Millipore) and then incubating with anti-UCP1 antibody (ab23841, Abcam, 1:250) overnight at 4 °C. The next day, slides were washed in PBS and were incubated with goat anti-rabbit immunoglobulin G (IgG) (H + L) secondary antibody conjugated with Alexa Fluor 594 (Invitrogen, 1:200). Nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole).
9
Quantifying Beige Adipocytes in Adipose Tissue
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Sections of mesenteric adipose tissue and inguinal adipose tissue were stained with anti-UCP1 antibody (diluted at 1:200, ab23841, Abcam, Cambridge, UK) and then stained with secondary antibody (anti-rabbit diluted 1:2, Dako EnVision® + system-HRP labelled polymer; Agilent Technologies, Santa Clara, CA). Photographs were taken of 5 random areas of each adipose tissue sample at 400 × magnification. The number of UCP1 positive cells among the total adipocytes (500–800 cells) was measured by cellSens, and the rates of UCP1 positive cells were calculated.
10
Immunohistochemical Analysis of Adipose Tissue
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Tissues were fixed with 10% formalin and embedded in paraffin for histological analysis. Deparaffinized tissue slides were stained with primary antibodies for uncoupling protein-1 (UCP1) (1:200; ab23841; Abcam, Cambridge, MA), CBP/p300-interacting transactivator 1 (CITED1) (1:300; ab15096; Abcam), MAC-2 (1:500; CL8942AP; Cedarlane Laboratories), and endomucin (1:200; sc-65495; Santa Cruz Biotechnology). Biotin-labeled secondary antibodies were used. The reaction was visualized by the DAB Chromogen A system (DakoCytomation, Carpinteria, CA) and counterstained with hematoxylin. Images were acquired using a Coolscope (Nikon, Tokyo, Japan). Hematoxylin and eosin (H&E) staining and Masson’s Trichrome C staining were performed by Dr. John Shelton at the University of Texas Southwestern Medical Center Histology Core.
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