All proteins were extracted with RIPA lysis buffer and protein concentrations were determined using the BCA assay (Pierce, Rockford, IL, USA). For mouse skin, the tissue was homogenized in liquid nitrogen and then lysed in RIPA lysis buffer. Equal amounts of protein were subjected to electrophoresis. Western blotting was performed as described previously (32 (link), 33 (link)). Antibodies used included COX-2, AKT, p53, p21, Chk1, Chk2, XPC, DDB1, DDB2, γH2AX, β-actin, GAPDH (Santa Cruz), SIRT6, p-AKT (serine 473), Cleaved-caspase3, p-AMPK, p-Chk1, p-Chk2 (Cell Signal), acetylated k382 p53 (ac-p53,Abcam), and ac-H3K9 (Sigma).
Protocol detail
Western Blot Analysis of Skin Proteins
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Actin
Antibodies
Assay
Buffer
Caspase3
Cell
Cox 2
Ddb1
Ddb2
Electrophoresis
Gapdh
Mouse
Nitrogen
Protein
Ripa
Serine
Sirt6
Skin
Tissue
Corresponding Organization :
Other organizations : University of Chicago, Indian Institute of Science Bangalore, National Institute of Diabetes and Digestive and Kidney Diseases
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Variable analysis
independent variables
- Protein extraction method (RIPA lysis buffer)
- Tissue homogenization (in liquid nitrogen)
- Protein concentration (BCA assay)
- Protein expression (Western blotting)
- Posttranslational modifications (phosphorylation, acetylation)
- Equal amounts of protein subjected to electrophoresis
- Western blotting protocol (as described previously)
- Positive controls: β-actin, GAPDH (loading controls)
- Negative control: Not explicitly mentioned
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