The protocol of Western blotting was based on that of a previously published study (14 (link)). The same amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were then blocked in 5% skim milk for at least 2 h and incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. Antibodies against PSMD12 (11412-1-AP; Proteintech, China), FLAG (F1804; Sigma, USA), Nrf2 (16396-1-AP; Proteintech, China), NRBP2 (21549-1-AP; Proteintech, China), p-Akt (4060; Cell Signaling Technology, USA), Akt (2920; Cell Signaling Technology, USA), p-mTOR (5536; Cell Signaling Technology, USA), mTOR (2983; Cell Signaling Technology, USA), and β-actin (A5441; Sigma, USA) were used.
Protocol detail
Western Blotting for Protein Analysis
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Actin
Antibodies
Membranes
Milk
Mtor
Nitrocellulose
Nrf2
Proteins
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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Variable analysis
independent variables
- Amounts of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- Expression levels of PSMD12, FLAG, Nrf2, NRBP2, p-Akt, Akt, p-mTOR, mTOR, and β-actin
- Blocking membranes in 5% skim milk for at least 2 h
- Incubating with primary antibodies at 4 °C overnight
- Incubating with secondary antibodies at room temperature for 1 h
- Antibodies against PSMD12, FLAG, Nrf2, NRBP2, p-Akt, Akt, p-mTOR, mTOR, and β-actin were used as positive controls to measure the expression levels of these proteins
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