Whole-cell extracts were prepared using lysis buffer (Cell Signaling Technology). Cell lysates (20 μg) were separated by SDS-PAGE, and blotting performed as previously described.26 (link) Rabbit anti-GSTA4 (Abnova; H00002941-D01P, 1:2,000), anti-Nrf2 (Cell Signaling Technology; #12721, 1:1,000), anti-phospho-c-Jun (Ser73) (Cell Signaling Technology; #3270, 1:1,000), and anti-phospho-c-Fos (Ser32) (Cell Signaling Technology; #5348, 1:1,000) were used as primary antibodies. Murine anti-β-actin loading control was purchased from Santa Cruz Biotechnology (sc-81178, 1:2,000). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (Life Technologies; #65–6120, 1:10,000) and horse anti-mouse IgG-HRP conjugate (Cell Signaling Technology; #7076, 1:5,000) were used as secondary antibodies. Signals were generated by Clarity™ Western ECL Substrate (Bio-Rad). Images were captured by ChemiDoc XRS+ system and analyzed using Image Lab software (Bio-Rad).
Protocol detail
Western Blot Analysis of Cell Signaling Proteins
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lysis buffer
anti-Nrf2
anti-phospho-c-Jun (Ser73)
anti-phospho-c-Fos (Ser32)
horse anti-mouse IgG-HRP conjugate
Clarity™ Western ECL Substrate
ChemiDoc XRS+ system
Image Lab software
Actin
Anti igg
Antibodies
Buffer
Cell
Cell extracts
Goat
Horse
Horseradish peroxidase
Mouse
Nrf2
Rabbit
Sds page
Corresponding Organization :
Other organizations : University of Oklahoma Health Sciences Center, Oklahoma City VA Health Care System
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Variable analysis
independent variables
- None explicitly mentioned
- Levels of GSTA4 protein
- Levels of Nrf2 protein
- Levels of phosphorylated c-Jun (Ser73)
- Levels of phosphorylated c-Fos (Ser32)
- Cell lysis buffer used (Cell Signaling Technology)
- Amount of cell lysate loaded (20 μg)
- SDS-PAGE separation and Western blotting technique used as previously described (reference 26)
- Primary antibodies used: Rabbit anti-GSTA4 (1:2,000), anti-Nrf2 (1:1,000), anti-phospho-c-Jun (Ser73) (1:1,000), and anti-phospho-c-Fos (Ser32) (1:1,000)
- Secondary antibodies used: Goat anti-rabbit IgG-HRP conjugate (1:10,000) and horse anti-mouse IgG-HRP conjugate (1:5,000)
- Loading control used: Murine anti-β-actin (1:2,000)
- None explicitly mentioned
- None explicitly mentioned
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