Cells were collected and lysed in NP-40 buffer. The bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA) was used to measure the protein content. Proteins were separated by using SDS-PAGE (8%) and transferred to nitrocellulose membranes 12 (link). The primary antibodies IDO (Thermo Scientific, Rockford, IL, USA), mTOR (Cell Signaling, Danvers, MA, USA), phosphorylation-mTOR (Cell Signaling), protein kinase B (AKT) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), phosphorylation-AKT (Santa Cruz Biotechnology, Inc.), p70S6K (Cell Signaling), phosphorylation-p70S6K (Cell Signaling), and β-actin (Sigma Aldrich)) were used to detect the protein expression. Anti-murine or -rabbit secondary antibodies were used to recognize primary antibodies. The enhanced chemiluminescence system was used to visualize the protein expressions and signals were quantified with ImageJ software 13 (link).
Protocol detail
Protein Expression Analysis in Cells
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BCA) protein assay
phosphorylation-mTOR
protein kinase B (AKT)
phosphorylation-AKT
p70S6K
phosphorylation-p70S6K
β-actin
Actin
Antibodies
Assay
Bicinchoninic acid
Buffer
Cells
Chemiluminescence
Membranes
Mtor
Murine
Nitrocellulose
Np 40
P70s6k
Phosphorylation
Protein
Protein kinase b
Rabbit
Sds page
Corresponding Organization : China Medical University Hospital
Other organizations : Kaohsiung Armed Forces General Hospital, Kaohsiung Medical University, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Chung-Ho Memorial Hospital
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Variable analysis
dependent variables
- Protein expression of IDO
- Protein expression of mTOR
- Phosphorylation of mTOR
- Protein expression of AKT
- Phosphorylation of AKT
- Protein expression of p70S6K
- Phosphorylation of p70S6K
- NP-40 buffer used for cell lysis
- BCA protein assay used to measure protein content
- SDS-PAGE (8%) used to separate proteins
- Nitrocellulose membranes used for protein transfer
- Primary antibodies used to detect protein expression: IDO, mTOR, phosphorylation-mTOR, AKT, phosphorylation-AKT, p70S6K, phosphorylation-p70S6K, β-actin
- Anti-murine or -rabbit secondary antibodies used to recognize primary antibodies
- Enhanced chemiluminescence system used to visualize protein expressions
- ImageJ software used to quantify protein expression signals
- β-actin used as a loading control
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