The total protein of mouse breast tissues and MAC-T cells was separated by RIPA reagent (Biosharp, China). The BCA Protein Assay Kit (Thermo Scientific, MA, USA) was performed to detect protein concentration. The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); then, polyvinylidene difluoride (PVDF) membranes were used to receive the transferred protein from the gel. The membranes were blocked with 5% skimmed milk for 2 h; then, the primary antibodies (BiP, Ero1-Lα, PDI, IRE1α, PERK, CHOP, JNK, ERK, p38, p65, and β-actin from Cell Signaling Technology, USA; PPARγ from Abcam, UK) were incubated at 4°C overnight, and then, the secondary antibody (anti-rabbit IgG and HRP-linked antibody from Cell Signaling Technology, USA) was incubated at 37°C for 1 h. Finally, the immunoblot signal was displayed with ECL ultrasensitive chemiluminescent solution with chemiluminescence imaging system.
Pparγ
Manufactured by Abcam
Sourced in United States, United Kingdom, China
PPARγ is a nuclear receptor that functions as a transcription factor. It plays a role in the regulation of genes involved in cell differentiation and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (17 mentions)
C ebpα, by Abcam (15 mentions)
Gapdh, by Abcam (13 mentions)
Pvdf membrane, by Merck Group (8 mentions)
β actin, by Cell Signaling Technology (6 mentions)
β actin, by Merck Group (6 mentions)
β actin, by Santa Cruz Biotechnology (6 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
C ebpβ, by Abcam (5 mentions)
Bcl 2, by Abcam (5 mentions)
Sirt1, by Abcam (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
C ebpα, by Cell Signaling Technology (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
P erk, by Cell Signaling Technology (3 mentions)
Oil red o, by Merck Group (3 mentions)
Fabp4, by Abcam (3 mentions)
Srebp1, by Abcam (3 mentions)
Ampkα, by Cell Signaling Technology (3 mentions)
115 protocols using pparγ
1
Protein Expression Analysis in Mouse Tissues
2
Chondrocyte Protein Expression Analysis
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Chondrocyte pellets were dissolved in citrate-EDTA buffer (55 mM/5 mM; pH 6.8) for 10 min at 37 °C and centrifuged at 10 K rpm for 15 s. Then, the cell pellet was washed five times with PBS and resuspended in 200 μL of SDS-NuPAGE buffer for NuPAGE gel (Invitrogen, Grand Island, NY, USA) running. Immunoblotting was performed using monoclonal antibodies against Type Ⅰ collagen, Type Ⅱ collagen (1:1000 dilution; EMD Millipore, Billerica, MA, USA), MMP-13 (Abcam, Cambridge, MA, USA) TGF-β (1:1000 mg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and PPAR-γ (Abcam, Cambridge, MA, USA). The loading control, β-actin, will be detected using anti-β-actin antibody (1:10,000 dilution; Abcam, Cambridge, MA, USA). Secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for TGF-β 1:20,000 dilution and mouse for type Ⅱ collagen and β-actin, 1:5000 dilution) was used. Proteins separated in the gel and the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm which was enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).
3
Western Blot Analysis of Lipid Metabolism
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Immunoreactive bands were visualized using HRP-conjugated secondary antibodies and subsequent ECL detection (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Mouse or rabbit monoclonal antibody for ROCK II (Upstate, Lake Placid, NY), RhoA (Santa Cruz Biotechnology, Santa Cruz, CA), HMG-CoA reductase (Upstate), PPARγ (Abcam, Cambridge, UK), ABCA1 (Cell Signaling, Boston, MA), ApoA-I (Abcam), LXRα (Santa Cruz Biotechnology), LDLR (Abcam), HSL (Cell Signaling), eNOS (Abcam), and the loading control protein β-actin (Sigma-Adrich) were used in our Western blot analyses. Rabbit polyclonal antibody was used to recognize both PPARγ1 and PPARγ2 in experiments. Rp-8-pCPT-cGMPS and C3 exoenzyme were purchased from Sigma-Adrich. HFD was a basal purified diet (W/60% energy from fat, Blue:58G9 Test Diet; Richmond, VA). LDL was purchased from Abcam. Oxidized LDL (oxLDL) was purchased from Biomedical Technologies Inc. (Stoughton, MA).
4
Western Blot Analysis of PPAR-γ Expression
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Treated or transfected cells were collected at indicated time points, followed by the extraction of whole protein using RIPA buffer (Beyotime Biotechnology, Shanghai, China). Then, equal weight (40 µg) protein in each sample was separated by SDS-PAGE electrophoresis and then transferred to NC membranes (Millipore, Bedford, MA, USA). After blocking in 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against PPAR-γ or β-actin (Abcam, Cambridge, UK) overnight at 4℃. Subsequently, the membranes were probed with horseradish peroxidase-conjugated appropriate secondary antibody for 1 h at room temperature. Finally, specific protein signals in the membranes were detected using Clarity™ ECL Western Blotting Substrates (Bio-Rad, Hercules, CA, USA) and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
5
Histological Analysis of Liver Tissue
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Paraffin-embedded tissues were sliced into 5 μm sections and stained with hematoxylin and eosin (H&E). Plasma membrane was stained with CellMask Orange (Thermo Fisher Scientific). Frozen liver sections were cut into 5 μm slices and stained with 0.3% Oil red O (Sigma-Aldrich) in 60% isopropanol or 0.25 mg/mL filipin. For immunohistochemistry (IHC), sections were incubated overnight at 4°C (1:200 dilution) in a humidified chamber with the following primary antibodies; ACOX1 (Cusabio), H3K4me3 (Abcam), NUDT7 (Youngin Frontier), PPARγ (Abcam), FABP4(Abcam), FASN (Cell signaling technology), PMP70 (Abcam), and SCD1 (Abcam). Positive staining was visualized using the Liquid DAB Substrate Chromogen System (Dako) combined with counterstaining with hematoxylin. Histological images were acquired with a light microscope (Leica).
6
Protein Isolation and Western Blot Analysis
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Protein isolation and western blot analysis were performed as described in literature (Wu et al., 2010 (link)). Briefly, protein samples were placed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. The membranes were blocking with non-fat dry milk for 1 h and incubated at room temperature for 2 h with p-LKB1, LKB1, p-AMPKα, AMPKα, p-AMPKβ, AMPKβ, p-ACC, ACC (Cell Signaling Technology, Boston, MA, USA) or SREBP-1, CYP2E1, GAPDH, Sirt1, PPARγ (Abcam, Cambridge, MA, USA) or caspase-1, β-actin, PPARα (Santa Cruz, CA, USA) or IL-1β (R&D Systems Europe Ltd., Abingdon, UK). Then the membranes were followed by incubated with HRP-conjugated secondary antibody for 1 h at room temperature and visualized by ECL Prime Western Blotting Detection Reagent (Bio-Rad, USA).
7
Immunohistochemical Analysis of Mammary and Tumor Tissues
8
Adipose Tissue RNA Extraction and Gene Expression Analysis
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Total RNA from human adipose tissues, mouse adipose tissues and cells were extracted using Trizol reagent (TAKARA). cDNA was synthesized using a PrimeScript RT Reagent Kit (TAKARA). Quantitative Real-Time PCR was performed on a Roche LightCycler 480 Real-Time PCR system. Mouse Arbp was used as internal control in cells and mouse adipose tissues, and human 18S was chosen as normalization control in human adipose tissues. Protein expressions were detected by western blot as previously reported (Cai et al. 2019 (link)). The primary antibodies were specific for Sterol Regulator Elementing Binding Protein 1c (SREBP1C) (Abclonal), adiponectin (CST), peroxisome proliferator-activated receptor γ (PPARγ) (CST), CCAAT-enhancer-binding protein α (C/EBPα) (Abcam), uncoupling protein 1 (UCP1) (Santa), PPARγ coactivator-1α (PGC1α) (Santa), Wnt Family Member 10B (Wnt10B) (Abclonal), SMAD Family Member 2 (SMAD2) (CST), phosphor-SMAD2 (CST), and β-ACTIN server as loading control.
9
Dual-labeling Immunohistochemistry and Sirius Red Staining
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Double fluorescence staining was performed with liver sections as we described previously 9 . Briefly, paraformaldehyde‐fixed liver sections were blocked with normal serum and incubated with primary antibody against 4‐hydroxynonenal (4‐HNE, 1:50; Abcam, Cambrige, MA, USA), Sonic hedgehog (Shh, 1:50; Santa Cruz), Ser 473 phosphorylated AKT (p‐AKT, 1:100; Cell Signaling Technology Inc, Bevely, MA, USA), β‐catenin (1:100; Santa Cruz), PPARγ (1:100, Abcam), GATA3 (1:50; Santa Cruz) and primary antibody against synaptophysin (SYP, 1:10; Abcam), a marker for quiescent and activated HSCs 21 , followed by incubation with DyLight594‐conjugated secondary antibody (1:500; ImmunoReagents, Inc, Raleigh, NC, USA) and DyLight488‐conjugated secondary antibody (1:500; ImmunoReagents, Inc). The images were captured with the fluorescence microscope and representative images were shown.
Sirius red was used to stain collagen on liver. Briefly, mouse liver sections were stained with picric acid‐fast green (Amresco, Solon, OH, USA) and then incubated with picric acid–sirius red (Amresco) for 1 hr. The images were captured with light microscope and representative images were shown (Data S2).
Sirius red was used to stain collagen on liver. Briefly, mouse liver sections were stained with picric acid‐fast green (Amresco, Solon, OH, USA) and then incubated with picric acid–sirius red (Amresco) for 1 hr. The images were captured with light microscope and representative images were shown (Data S2).
10
Western Blot Analysis of Cell Signaling
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The samples were lysed in RIPA buffer supplemented with protease inhibitors and subjected to ultrasonics at low frequency. The supernatant containing total proteins was harvested after centrifugation and the protein concentration was measured by bicinchoninic acid protein assay (Beyotime, China). Equal amounts (30 μg) of total proteins were loaded on 10% or 15% SDS-PAGE according to the molecular weight of the target proteins. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore), and blocked in 5% BSA for 2 h at room temperature, followed by incubation overnight at 4°C with primary antibodies for LC3 (Cell Signaling Technology, USA), Beclin1 (Cell Signaling Technology, USA), ATG5 (Cell Signaling Technology, USA), P62 (Proteintech, USA), ALP (R&D systems, USA), Runx2 (Cell Signaling Technology, USA), PPAR-γ (Abcam, UK), OPG (Abcam, UK), RANKL (Abcam, UK) and Actin (Abcam, UK). After rinsed with tris buffered saline supplemented with tween (TBST) three times, membranes were incubated with secondary antibodies (Cowin Biotech Co, Beijing, China) for 1 h at room temperature. Blots were captured with a Western-Light Chemiluminescent Detection System (Tanon, Shanghai, China).
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