Western blots were performed as described previously9 (link). Briefly, the rats' left ventricle tissue and NRC samples were lysed in a lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.25% superoxide dismutase, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO3, 1 mM phenylmethylsulphonyl fluoride, and a proteinase inhibitor cocktail (Roche). The samples were evaluated by SDS-PAGE. Briefly, total protein from the samples was determined before being subjected to polyacrylamide gel electrophoresis and being transferred to a nitrocellulose (NC) membrane. The NC membrane was immunoblotted with an anti-14-3-3 antibody (Santa Cruz; 1:1,000), LC3 antibody (Santa Cruz; 1:1,000), p62 antibody (Santa Cruz; 1:1,000), and anti-β-actin antibody (Santa Cruz; 1:10,000). β-Actin protein served as a loading control. Protein bands were evaluated by densitometry using the Odyssey infrared imaging system (LI-COR).
Protocol detail
Evaluating Protein Expression in Rat Ventricle
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anti-14-3-3 antibody
LC3 antibody
p62 antibody
anti-β-actin antibody
Odyssey infrared imaging system
14 3 3
Actin
Anti antibody
Antibody
Buffer
Densitometry
Edta
Left ventricle
Membrane
Nacl
Nitrocellulose
Nonidet p 40
Phenylmethylsulphonyl fluoride
Polyacrylamide gel electrophoresis
Protein
Proteinase inhibitor
Rats
Sds page
Superoxide dismutase
Tissue
Tris
Western blots
Corresponding Organization :
Other organizations : Air Force Medical University, Beijing Chest Hospital, Capital Medical University, Second Affiliated Hospital of Xi'an Jiaotong University
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Variable analysis
independent variables
- Left ventricle tissue
- NRC (neonatal rat cardiomyocytes) samples
- Protein expression levels of 14-3-3, LC3, p62
- Lysis buffer composition (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.25% superoxide dismutase, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO3, 1 mM phenylmethylsulphonyl fluoride, and a proteinase inhibitor cocktail)
- SDS-PAGE and transfer to nitrocellulose membrane
- Antibodies used for immunoblotting (anti-14-3-3, LC3, p62, and anti-β-actin)
- β-Actin protein as a loading control
- Densitometry analysis using Odyssey infrared imaging system
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