Cell lysates were prepared using complete lysis buffer (EMD Millipore, San Diego, CA) with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN). Protein quantification was performed using DC protein assay (Bio-Rad, Hercules, CA. Western blot analysis was performed as described previously (Goc, Al-Azayzih et al. 2013 (link), Gao, Al-Azayzih et al. 2015 (link)). Antibodies used include Src Tyr416, total-Src, Akt Ser473, total-Akt (Cell Signaling, Danvers, MA) and anti- β-actin (Sigma, St. Louis, MO). Densitometry was done using NIH Image J software.
Protocol detail
Western Blot Analysis of Signaling Proteins
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complete lysis buffer
protease and phosphatase inhibitor cocktails
DC protein assay
total-Src
Akt Ser473
total-Akt
anti- β-actin
Actin
Antibodies
Assay
Buffer
Cell
Densitometry
Diagnostics
Phosphatase
Protease
Protein
Western blot analysis
Corresponding Organization : Augusta University Health
Other organizations : Chongqing Medical University, Chongqing University, First Affiliated Hospital of Chongqing Medical University
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Variable analysis
independent variables
- None explicitly mentioned
- Src Tyr416 protein levels
- Total Src protein levels
- Akt Ser473 protein levels
- Total Akt protein levels
- Cell lysates prepared using complete lysis buffer with protease and phosphatase inhibitor cocktails
- Protein quantification performed using DC protein assay
- Western blot analysis performed as described previously
- Anti-β-actin antibody used as a loading control
- Positive control: Not specified
- Negative control: Not specified
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