Cultured cells or tissues were prepared with the T-PER tissue protein extraction reagent (2% SDS and 60 mM Tris HCl, pH 6.8) with the cocktail of proteinase inhibitors (Roche, Indianapolis, IN, USA) in it. The total protein tissue or cultured cells were homogenized in lysis buffer and were loaded onto the gel (20–40 μg) for electrophoresis. Proteins then were transferred onto 10% SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) and were then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The electroblotted membranes were blocked by TBS containing 5% non-fat milk (Santa Cruz, Dallas, TX, USA) and were probed with primary antibodies overnight at 4°C and immunoblotted with specific antibodies. Primary antibodies against the following proteins were used: UCP1, PGC1α, PRDM16, Cidea (Abcam, Cambridge, UK), PPARγ (CST, Danvers, MA, USA), 422/Ap2 (Santa Cruz Biotechnology), RPS3A (ProteinTech Group, Rosemont, IL, USA), Cyto c (CST), Actin (Sigma, St. Louis, USA).
Cidea
Manufactured by Abcam
Sourced in United Kingdom, United States
Cidea is a protein that is involved in the regulation of energy metabolism and thermogenesis. It is expressed in adipose tissue and plays a role in the control of energy expenditure and body weight. Cidea is a member of the cell death-inducing DFFA-like effector (CIDE) family of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cocktail of proteinase inhibitors, by Roche (1 mentions)
Rps3a, by Proteintech (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemiluminescent peroxidase substrate, by Merck Group (1 mentions)
Prdm16, by Abcam (1 mentions)
Pvdf nitrocellulose membrane, by Merck Group (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Quality one software, by Bio-Rad (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Chemigenius bioimaging system, by Syngene (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Pparγ, by Abcam (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Genesnap software, by Syngene (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
5 protocols using cidea
1
Protein Expression Analysis in Cultured Cells
2
Western Blot Analysis of Protein Targets
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Cells were lysed in RIPA buffer for 40 min at 4 °C centrifuge. Removing insoluble material by centrifugation at 12,000× g for 15 min at 4 °C, and the supernatants were used to assay protein levels. Protein samples (50 μg) were separated by electrophoresis on 12% and 5% SDS-PAGE gels using slab gel apparatus and then transferred to PVDF nitrocellulose membranes (Millipore, Boston, MA, USA) blocked with 5% Skim Milk Powder/Tween 20/TBST at room temperature for 2 h. Primer antibodies against Mark4 and GAPDH were purchased from Bioworld (MN, USA). Antibodies against LC3B-ll and LC3l, P62, UCP1, PGC1α, Prdm16, and Cidea were from Abcam (Cambridge, MA, USA). Additionally, antibodies against AMPK, phospho-AMPK, AKT, phospho-AKT, mTOR, and phospho-mTOR were from Cell Signaling (Danvers, MA, USA). Membranes were incubated with primary antibodies at 4 °C overnight and then incubated with the appropriate HRP-conjugated secondary antibodies (Boaoshen, China) for 2 h at room temperature. Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, Shanghai, China) and quantitative analysis of immune-blotted bands was performed using Quality One software (Bio-Rad).
3
Western Blot Analysis of CIDEA Regulation
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Western blot analysis was performed with whole lysates, cytosolic and nuclear protein extracts of cells transfected with CIDEA overexpressing plasmid or treated with inhibitors of PPARγ, HIF-1α or JNK as described,33 (link) using antibodies against CIDEA (Abcam, Cambridge, UK), PPARγ, HIF-1α (Novus Biological, Cambridge, UK), phospho-JNK, JNK, phospho-cofilin, Cofilin, phospho-VASP, VASP, phospho-STAT3 (Y705), STAT3, p53, acetyl-p53 (Lys-373 and Lys-382) (Millipore, Billerica, MA, USA); Cyclin B1, β-tubulin, GAPDH, C-23 and NFκB Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies were purchased from Cell Signaling (Danvers, MA, USA) unless otherwise mentioned. After addition of horse radish peroxidase-conjugated secondary antibodies (Vector Laboratories Inc., Burlingame, CA, USA) blots were exposed to Chemigenius Bioimaging System (Syngene, Cambridge, UK) and images were developed by Gene snap software (Syngene). Reprobing of the blots was performed after stripping to determine the loading control with anti-β-tubulin, GAPDH or C23 antibodies.
4
Western Blot Analysis of Thermogenic Proteins
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Proteins in SAT were extracted by lysing in modified radioimmunoprecipitation buffer (Thermo Fisher, Waltham, MA, USA), as previously described [19 (link)]. An equal amount of protein was loaded and separated using electrophoresis gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinylidene fluoride membranes. Blocker™ FL Blocking Buffer (Thermo Fisher, Waltham, MA, USA) was used to block the membrane along with primary antibodies for TBP (1:1000, Cell Signaling, Danvers, MA, USA) as a housekeeping control and UCP1 (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), CIDEA (1:1000, Abcam, Cambridge, MA, USA) and FGF21 (1:1000, Abcam, Cambridge, MA, USA) as target genes. Rabbit polyclonal antibody was used as a secondary antibody (1:25,000). Blots were developed using Li-COR Imager System (Lincoln, NE, USA).
5
Protein Extraction and Western Blot Analysis
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The cells were collected and washed twice with cold phosphate-buffered saline (PBS). Total cellular proteins were extracted using a protein extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). Protein Table 1. The primers sequences used for cDNA generation Table 2. Regression coefficients (R 2 ), amplification efficiency [E = (10 -1/slope -1) ×100%] and slope of the standard curves of the primer sets Tween-20 (TBS-T), supplemented with 3% BSA at room temperature for 4 h. The membranes were incubated with antibodies at 4°C overnight. Next, the membranes were washed with TBS-T for three times, followed by incubation with secondary antibody conjugated peroxidase for 45min at room temperature. Then, the membranes were washed four times for 5min and an immunodetection analysis was performed using an enhanced chemiluminescence solution (ECL, Pierce Biotechnology Inc., Chicago, IL, USA). Antibodies against Cidea (Abcam, Cambridge, UK) diluted at 1:500 in TBS-T, SREBP1 (Novus, USA) diluted at 1:1000 in TBS-T, FAS (CST, Danvers, USA) diluted at 1:500 in TBS-T, ACC (Abcam, Cambridge, UK) diluted at 1:1000 in TBS-T, β-actin (Santa Cruz, USA) diluted at 1:1000 in TBS-T. HRP-conjugated goat, anti-mouse or anti-rabbit secondary antibody (Boster, Wuhan, China) diluted at 1:5000 in TBS-T. Protein grey intensity was quantified by the Gel-Pro Analyzer program normalized to β-actin levels.
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