Western blot was performed51 (link) to measure the protein expression levels of DNMTs in the PFC. Primary antibodies were rabbit DNMT1 (1:1000, #5032; Cell Signaling Technology), DNMT3a (1:1000, #2160; Cell Signaling Technology), DNMT3b (1:2000, NB300-516; Novusbio, Littleton, CO), brain-derived neurotrophic factor (1:500, sc-546; Santa Cruz Biotechnology), and mouse β-actin (1:10,000, A1978; Sigma-Aldrich). The membranes were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000; Pierce, Rockford, IL) and anti-mouse antibody (1:30,000 for β-actin, Pierce). Signals were visualized using a chemiluminescence kit (Super Signal West Pico; Pierce) and intensity was measured by densitometry.
Protocol detail
Quantifying DNMT Protein Expression in PFC
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Actin
Anti antibody
Antibodies
Brain derived neurotrophic factor
Chemiluminescence
Densitometry
Dnmt1
Dnmt3b
Dnmts
Goat
Horseradish peroxidase
Membranes
Mouse
Protein
Rabbit
Western blot
Corresponding Organization : Kyung Hee University
Other organizations : Dongguk University, Korea Institute of Oriental Medicine, Chonnam National University
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Variable analysis
independent variables
- Protein expression levels of DNMTs (DNMT1, DNMT3a, DNMT3b)
- Protein expression levels of brain-derived neurotrophic factor (BDNF)
- β-actin expression level
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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