Total proteins were extracted using RIPA lysis buffer within 1% protease inhibitor cocktail (Millipore, Bedford, MA, USA) 0.1 mM according to the manufacturer’s instructions. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking, the membranes were then incubated at 4 °C overnight with the following primary antibodies: anti-HIF-1α, anti-VEGF-A, anti-JNK, anti-PPAR-γ, anti-C/EBP-α, anti-FABP4 (1:1000, all Cell Signaling Technology, Danvers, MA, USA), anti-NF-κB p65, anti-p-IκB, anti-TNF-α, anti-MCP-1 (1:1000, all Abcam, Cambridge, MA, USA), anti-SFRP5, anti-Wnt5a, anti-Wnt10b, anti-β-catenin (1:1000), and anti-β-actin (1:5000, all GeneTex, Irvine, CA, USA). After washing, the membranes were probed with corresponding second antibodies (1:3000, GeneTex). The density of the individual protein bands was quantified by densitometric scanning of the blots using ImageJ software.
Anti c ebpα
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-C/EBPα is a laboratory reagent used to detect the expression of the C/EBPα (CCAAT/enhancer-binding protein alpha) transcription factor in biological samples. C/EBPα plays a crucial role in the regulation of cell differentiation and proliferation. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to analyze the presence and distribution of C/EBPα in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti pparγ, by Cell Signaling Technology (14 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti fabp4, by Cell Signaling Technology (4 mentions)
Anti ampk, by Cell Signaling Technology (4 mentions)
Anti c ebpβ, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Anti adiponectin, by Cell Signaling Technology (2 mentions)
Anti glut4, by Cell Signaling Technology (2 mentions)
Anti β actin, by Boster Bio (2 mentions)
Anti hsl, by Cell Signaling Technology (2 mentions)
Insulin, by Merck Group (2 mentions)
Anti pi3k, by Cell Signaling Technology (2 mentions)
Anti c ebpβ, by Abcam (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Anti c ebpβ sc 150, by Santa Cruz Biotechnology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti fas, by Cell Signaling Technology (2 mentions)
Anti ap2, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
26 protocols using anti c ebpα
1
Western Blot Analysis of Adipogenic Markers
2
Berberine's Effects on Adipocyte Differentiation
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Dulbecco's modified Eagle's medium (DMEM) and other culture reagents were obtained from Gibco Life Technologies (Grand Island, NY).The cell culture plates were purchased from Nalge Nunc International (Roskilde, Denmark). Human insulin (HumulinR) was from Eli Lilly S.A.S.(Fegersheim, France). Bovine serum albumin (BSA), forskolin, IBMX, and dexamethasone were purchased from Sigma (St Louis, MO, USA). Compound C was purchased from Calbiochem (San Diego, CA). Anti-CREB, anti-phospho-CREB (Ser133), anti-PPARγ, anti-C/EBPα, anti-fatty acid synthase (FAS), anti-fatty acid binding protein 4 (FABP4), anti-C/EBPβ, anti-AMPK, anti-phospho-AMPK (Thr172), anti-acetyl-CoA carboxylase (ACC), anti-phospho-ACC(Ser79), anti-β-actin, anti-α1-tubulin, anti-mouse IgG and anti-rabbit IgG conjugated with horseradish peroxidase were from Cell Signaling Technology (Beverly, MA, USA). Murine-derived 3T3-L1 preadipocytes were purchased from American Type Culture Collection (Rockville, MD). Berberine was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).
3
Protein Extraction and Western Blot Analysis
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Proteins were collected on ice using RIPA (Beyotime, Shanghai, China) with phosphatase inhibitor (Roche Applied Science, Baden-Württemberg, Germany) and protease inhibitor (PMSF; Roche Applied Science). The concentration of protein was measured with a BCA kit (Biosharp, Anhui, China). 30 μg of protein were electrophoresed on 8%-10% SDS-polyacrylamide gel and transferred onto methanol-treated PVDF membrane (Millipore, MA, USA). The membranes were blocked with 5% non-fat milk for over 1 h and incubated with anti-IRX5 (Sigma-Aldrich, SAB1404807, MO, USA), anti-PGC-1α (Santa Cruz, SC-517380, Texas, USA), anti-GLUT1 (Abcam, ab115730, Cambridge, UK), anti-PPAR-γ (Abcam, ab178806, Cambridge, UK), anti-CEBP-α (Cell Signaling Technology, D56F10, Cambridge, UK), anti-β-actin (BioPM, PMK081W, Hubei, China), anti-β-Tubulin (Abmart, M20005F, Shanghai, China) antibodies at 4 °C overnight. An ECL system (Millipore) was used to visualize the target bands. Image J software (National Institutes of Health, Maryland, USA) was used for quantification by densitometry.
4
Western Blot Analysis of Cell Protein
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Proteins were extracted from cultured cells and VAT using RIPA lysis buffer. The protein concentration was determined with a bicinchoninic acid protein assay kit (Sigma). Proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes. After blocking for 1 h, the membranes were incubated with primary antibody at 4 °C overnight. The following antibodies were used: anti-Wnt5β (1:000; Abcam, Cambridge, UK), anti-C/EBP-α and anti-PPAR-γ (1:000; Cell Signaling Technology, Danvers, MA, USA). Membranes were incubated with the appropriate HRP-conjugated secondary antibody at room temperature for 2 h. The immunoreactive bands were visualized using ECL and normalized to GAPDH (the internal control).
5
Analyzing STMN1, E2F1, and C/EBPα in DENV2-infected Endothelial Cells
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Lysates from EAhy926, EAhy-223-down, EAhy-223-up, EAhy-STMN1-down, EAhy-STMN1-up, and control cells with or without DENV2 infection were obtained using RIPA lysis buffer. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, USA). Subsequently, anti-STMN1 (1:1000 dilution; BD, USA), anti-E2F1 (1:400 dilution; Santa Cruz Biotechnology, Inc., USA), anti-C/EBPα (1:1000 dilution; Cell Signaling Technology, USA) or anti-NS1 (1:1000 dilution; gift of Professor X.Y. Che, China) was used as the primary antibody. GAPDH was detected with an anti-GAPDH polyclonal antibody (1:1000; Beijing Protein Innovation, China) to demonstrate equal loading of protein samples. After incubation with a secondary antibody conjugated to horseradish peroxidase, the protein bands were incubated with ECL™ Western Blot Detection Reagents (GE Healthcare, USA), captured on film, and analyzed with the Quantity one software.
6
Adipogenic Differentiation of Rat MSCs
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Mesenchymal stromal cells (MSCs) from 12-week old WT and Cyp2j4−/− rats were obtained as previously described [32] (link). MSCs cells were allowed to grow in Supplemented MesenCult™ MSC Medium (STEMCELL Technologies, UK) for 5 days on Petri dishes (Nunc, ThermoFisher Scientific, UK). MSCs from WT and Cyp2j4−/− rats were differentiated into mature adipocytes by incubation with an adipogenic induction medium (StemPro®,Gibco, UK) for 14 days.
Antibodies used in western blot were: anti-PPAR-γ (C26 H12, Cell Signaling #2435, 1:1000), anti CEBP-α (Cell Signaling #2295, 1:1000), anti-Phospho-Akt-Ser473 (D9E, Cell Signaling #4060, 1:2000), anti-Phospho-Akt-Thr308 (244F9, Cell Signaling #4056, 1:1000), anti-panAkt (C67E7, Cell Signaling #4691, 1:1000) and anti-β-Actin Antibody (C4, sc-47778, 1:10,000), anti-PPARα (H2, SC-398,394, 1:1000), anti-PPARβ/δ (F-10, SC-74517, 1:1000), anti-FXR (D-3, SC-25309, 1:1000), anti-LXRα (ab2585, 1:1000), and anti-β-Actin Antibody (C4, sc-47778, 1:10,000).
Antibodies used in western blot were: anti-PPAR-γ (C26 H12, Cell Signaling #2435, 1:1000), anti CEBP-α (Cell Signaling #2295, 1:1000), anti-Phospho-Akt-Ser473 (D9E, Cell Signaling #4060, 1:2000), anti-Phospho-Akt-Thr308 (244F9, Cell Signaling #4056, 1:1000), anti-panAkt (C67E7, Cell Signaling #4691, 1:1000) and anti-β-Actin Antibody (C4, sc-47778, 1:10,000), anti-PPARα (H2, SC-398,394, 1:1000), anti-PPARβ/δ (F-10, SC-74517, 1:1000), anti-FXR (D-3, SC-25309, 1:1000), anti-LXRα (ab2585, 1:1000), and anti-β-Actin Antibody (C4, sc-47778, 1:10,000).
7
Adipogenic and Epithelial-Mesenchymal Transition Modulation
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3T3-L1 cells were purchased from the American Type Culture Collection (USA). 3T3-L1 cells were maintained in DMEM (Thermo Fisher Scientific, USA), bovine calf serum (BCS, HyClone, USA) and 1% penicillin/streptomycin (Corning Inc., USA). MDA-MB-231 and MTV/TM-011 cells were obtained from Korean Cell Line Bank (Republic of Korea). MDA-MB-231 and MTV/TM-011 cells were cultured in RPMI (Corning Inc.) containing 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Corning Inc.). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2/95% air. Recombinant mouse GREM2 protein was obtained from R&D Systems (USA). Recombinant mouse IL-6 protein was purchased from Sino Biological Inc. (China). Rabbit polyclonal GREM2 antibody was purchased from Abcam (UK). Anti-PPARγ, anti-C/EBPα, anti-FABP4, anti-vimentin, anti-slug, anti-twist1, and anti-β-actin were obtained from Cell Signaling Technology (USA). Epigallocatechin gallate (EGCG), metformin, and PKH26/PKH67 fluorescent cell linker kits were purchased from Sigma-Aldrich (USA).
8
Adipokine Secretion in Adipogenesis
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Cells and tissue extracts were prepared as previously described and 30 μg of protein extracts were loaded for SDS-PAGE [22] (link). The antibodies used were: anti-MR (1/100; kindly provided by Gomez-Sanchez CE), anti-GR (1/1000 Santa Cruz), anti-SGK1 (1/1000 Sigma), anti-HSP90 (1/1000 Cell Signaling), anti-PPARγ (1/500 Cell Signaling), anti-CEBPα (1/500 Cell Signaling) and anti-actin (1/1000 Sigma).
For the adipokine array, cell supernatants were assessed following manufacturer's instructions (Mouse Adipokines Array Kit, R&D System). Briefly, supernatants from 6 independent experiments were collected and pooled in 2 aliquots (pre-adipocytes and mature adipocytes). 300 μl of the collected medium was incubated over-night with the adipokine antibody cocktail and the blocked nitrocellulose membrane. After several washes, detection was performed using a chemi-luminescence kit and autoradiography films. Quantification of the immunoblots was performed using ImageJ software (NIH).
For the adipokine array, cell supernatants were assessed following manufacturer's instructions (Mouse Adipokines Array Kit, R&D System). Briefly, supernatants from 6 independent experiments were collected and pooled in 2 aliquots (pre-adipocytes and mature adipocytes). 300 μl of the collected medium was incubated over-night with the adipokine antibody cocktail and the blocked nitrocellulose membrane. After several washes, detection was performed using a chemi-luminescence kit and autoradiography films. Quantification of the immunoblots was performed using ImageJ software (NIH).
9
Quantification of Glucose Uptake Proteins
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The expression levels of glucose uptake-related proteins were measured by Western blotting, as described previously [38 (link)]. Briefly, 3T3-L1 cells were lysed using radioimmunoprecipitation assay buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM ethylenediaminetetraacetic acid, 10 mM NaF, 2 mM NaVO4, and protease inhibitor cocktail; Roche Korea, Seoul, Korea). Total proteins were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Merck KGaA, Darmstadt, Germany) and blotted with the indicated antibodies. The membranes were treated with an anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, USA), followed by detection using enhanced chemiluminescence (ECL) reagents (GE Healthcare Korea, Songdo, Korea) in a WSE-6200 LuminoGraph II (ATTO, Tokyo, Japan). Anti-PPARγ, anti-C/EBPα, anti-adiponectin, and aP2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
10
Circadian Rhythm Protein Analysis
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The cells were lysed with RIPA lysis buffer (Millipore-Sigma, Billerica, MA, USA) containing protease inhibitors (Millipore-Sigma). Protein concentration was determined by BCA assays, and 30 µg cell lysates were resolved by SDS-PAGE on 4–12% gradient Bio-Tris gels, transferred to nitrocellulose membranes (Thermo Fisher Scientific), and probed with each of the following antibodies: anti-CLOCK, anti-BMAL1, anti-TIMP3, anti-C/EBP-α, anti-C/EBP-β, anti-TNF-α, anti-NF-κB, anti-phospho-NF-κB, and anti-I-κB (Cell Signaling Technology, Beverly, MA, USA); anti-MMP-1 (courteously provided by Prof. Jin Ho Chung, Seoul National University College of Medicine, Seoul, South Korea); and GAPDH and horseradish peroxidase–conjugated secondary anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Western blotting luminol reagent (Santa Cruz Biotechnology) was used to develop the signals. For zymogram analysis, the cultured medium was harvested every 4 or 12 h after synchronization or UV irradiation and loaded on 10% Zymogram (gelatin) Protein Gels (Thermo Fisher Scientific). The gels were incubated at 37°C overnight and stained with 0.5% Coomassie blue (Millipore-Sigma) according to the manufacturer’s instructions.
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