Adipose tissues were homogenized and the protein concentration was determined by Bradford assay as previously described [20 (link)]. The proteins were separated on 4–12% acrylamide SDS/PAGE gels (Bio-Rad, Hercules, CA) to evaluate the expression of H6PDH (Cat# sc-67394, Santa Cruz, Dallas, Texas), 11β-HSDl (Cat# sc-20175, Santa Cruz, Dallas, Texas), ACC (Cat# C83B10, Cell Signaling, Danvers, MA), ACL (Cat# 4332, Cell Signaling, Danvers, MA), pSer79 ACC (Cat# D7D11, Cell Signaling, Danvers, MA), pSer455 ACL (Cat# 4331, Cell Signaling, Danvers, MA), total HSL (Cat# 4107S, Cell Signaling, Danvers, MA), phospho-Ser660 HSL(Cat# 4126S Cell Signaling, Danvers, MA), ATGL (Cat# 2138S, Cell Signaling, Danvers, MA), and β-actin (Cat# 3700S, Cell Signaling, Danvers, MA). Adipose proteins were transferred to nitrocellulose membranes and incubated with the appropriate primary and secondary antibodies. Protein bands were visualized using ECL (Thermo Fisher Scientific Inc. Waltham, MA) and quantified by Eagle Eye II Quantitation System (Stratagene, CA, U.S.A.).
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Adipose Tissue Protein Expression Analysis
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Acrylamide
Actin
Adipose
Adipose tissues
Antibodies
Assay
Eagle
Gels
Membranes
Nitrocellulose
Protein
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Corresponding Organization : Charles R. Drew University of Medicine and Science
Other organizations : First Hospital of Jilin University, UCLA Medical Center, Harbor–UCLA Medical Center, Shanghai Sixth People's Hospital, Western University of Health Sciences
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Variable analysis
independent variables
- Adipose tissues were homogenized
- Protein concentration determined by Bradford assay
- Expression of H6PDH, 11β-HSD1, ACC, ACL, pSer79 ACC, pSer455 ACL, total HSL, phospho-Ser660 HSL, ATGL, and β-actin
- Protein separation on 4–12% acrylamide SDS/PAGE gels
- Protein transfer to nitrocellulose membranes
- Incubation with appropriate primary and secondary antibodies
- Protein band visualization using ECL and quantification by Eagle Eye II Quantitation System
- No positive or negative controls were explicitly mentioned in the input protocol.
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