Western Blot Analysis of AKT-mTOR Pathway

An exact weight of 0.2 g brain tissue was immersed once in 0.1 M ice-cold phosphate-buffered saline (BPS). After air drying, the tissue was placed in 500 μl ice-cold RIPA lysis buffer with protease inhibitors to prepare the tissue homogenates. After 30 minutes on ice, the homogenates were centrifuged for 5 min (12,000 rpm, 4 °C), and the supernatants were collected to be used as sample solutions for western blot analysis, as described in a previous study31 (link). Adequate sample solutions were removed to measure the total protein concentrations using the bicinchoninic acid (BCA) method. The membranes were incubated with the following primary antibodies: AKT (1:2,000; Cell Signaling Technology, Boston), phospho-AKT (1:2,000; Cell Signaling Technology, Boston), mTOR (1:1,000; Cell Signaling Technology, Boston), phospho-mTOR (1:100; Cell Signaling Technology, Boston), phospho-p70S6K (1:200; Santa Cruz Biotechnology, California), p70S6K (1:1,000; Cell Signaling Technology, Boston) and β-actin (1:4,000; Santa Cruz Biotechnology, California). The band density was quantified using Image J software. The amount of protein expression is presented relative to the levels of β-actin.

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Xing Z., Xia Z., Peng W., Li J., Zhang C., Fu C., Tang T., Luo J., Zou Y., Fan R., Liu W., Xiong X., Huang W., Sheng C., Gan P, & Wang Y. (2016). Xuefu Zhuyu decoction, a traditional Chinese medicine, provides neuroprotection in a rat model of traumatic brain injury via an anti-inflammatory pathway. Scientific Reports, 6, 20040.

Publication 2016

phospho-AKT phospho-mTOR phospho-p70S6K p70S6K β-actin

Actin Antibodies Bicinchoninic acid Brain 2 Buffer Cold Membranes Mtor P70s6k Phosphate Protease inhibitors Protein Ripa Saline Tissue Western blot analysis

Corresponding Organization :

Other organizations : Xiangya Hospital Central South University, Central South University, Shaoyang University

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Variable analysis

independent variables

Immersion of brain tissue in 0.1 M ice-cold phosphate-buffered saline (PBS)

dependent variables

Protein expression levels of AKT, phospho-AKT, mTOR, phospho-mTOR, phospho-p70S6K, and p70S6K

control variables

Weight of brain tissue (0.2 g)
Temperature (ice-cold)
Time of incubation in RIPA lysis buffer (30 minutes)
Centrifugation conditions (12,000 rpm, 4 °C, 5 minutes)
Primary antibody concentrations (AKT 1:2,000, phospho-AKT 1:2,000, mTOR 1:1,000, phospho-mTOR 1:100, phospho-p70S6K 1:200, p70S6K 1:1,000, β-actin 1:4,000)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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