Whole-cell lysates from frozen tissues and brown adipocytes were isolated using radioimmunoprecipitation assay (RIPA) lysis buffer (150 mmol/L Tris-HCl, 50 mmol/L NaCl, 1% NP-40, 0.1% Tween-20). Protease and phosphatase inhibitors were added to all buffers before experiments. Western blot was performed as previously described [21 (link)]. Protein concentrations were assayed using a Quick StartTM Bradford Assay (Bio-Rad, Hercules, CA, USA). The primary antibodies anti-phosphorylated AMPK (p-AMPK) antibody (Cell Signaling, Danvers, MA, USA), anti-PPARγ antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGC-1α, and anti-UCP1 antibody (Abcam, Cambridge, UK) were incubated overnight at 4°C, and specific proteins were visualized by the WESTSAVEupTM Detection system (AbFrontier, Seoul, Korea). Band intensities were measured using the GeneTool (SynGene, Cambridge, UK) and normalized to β-actin.
Protocol detail
Protein Expression Analysis in Brown Adipocytes
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Actin
Anti antibody
Antibodies
Assay
Brown adipocytes
Buffers
Cell
Frozen
Inhibitors
Nacl
Np 40
Pgc 1α
Phosphatase
Pparγ
Protease
Proteins
Radioimmunoprecipitation assay
Tissues
Tris
Tween 20
Ucp1
Western blot
Corresponding Organization :
Other organizations : Yonsei University
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Variable analysis
independent variables
- None explicitly mentioned
- Phosphorylation of AMPK (p-AMPK)
- Levels of PPARγ
- Levels of PGC-1α
- Levels of UCP1
- Whole-cell lysates from frozen tissues and brown adipocytes
- RIPA lysis buffer (150 mmol/L Tris-HCl, 50 mmol/L NaCl, 1% NP-40, 0.1% Tween-20)
- Protease and phosphatase inhibitors added to all buffers
- Protein concentrations assayed using a Quick Start™ Bradford Assay (Bio-Rad, Hercules, CA, USA)
- Primary antibodies: anti-phosphorylated AMPK (p-AMPK) antibody (Cell Signaling, Danvers, MA, USA), anti-PPARγ antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGC-1α, and anti-UCP1 antibody (Abcam, Cambridge, UK)
- Primary antibodies incubated overnight at 4°C
- Specific proteins visualized by the WESTSAVEup™ Detection system (AbFrontier, Seoul, Korea)
- Band intensities measured using the GeneTool (SynGene, Cambridge, UK) and normalized to β-actin
- No positive or negative controls were explicitly mentioned in the input.
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