These analyses were performed for MAFbx/Atrogin-1, MuRF1 and Gapdh mRNAs in plantaris muscles as described23 (link). Real-time PCR was performed using primers for MAFbox/Atrogin 1 and 18S rRNA from Applied Biosystems (Foster City, CA). Immunoblot analysis—Immunoblot analysis was performed as described6 (link) with the following primary antibodies: CuZnSOD (Abcam), MnSOD (Abcam), EcSOD (R&D, Minneapolis), peroxisome proliferator-activated receptor γ coactivator 1-α (PGC- 1α) (Chemicon/Millipore), cytochrome oxidase IV (COX IV) (Invitrogen), cytochrome C (Cyt C) (Cell Signaling, Danvers), malondialdehyde (MDA) (Academy Biomedical Company, Inc., Houston) and 4-hydroxynonenal (4-HNE) (ab48506, Abcam). Hybridoma for antibodies against MHC I (BA-F8), MHC IIa (SC-71) and MHC IIb (BF-F3) were purchased from German Collection of Microorganisms and Cell Cultures. All the bands for analysis have been validated previously3 (link), 6 (link), 24 (link). OxyBlot Protein Oxidative Detection Kit (Millipore) was used for immunblot detection of carbonylated proteins. Immunoblots were analyzed by Odyssey Infrared Imaging System (LI-COR Biosciences, NE) and quantified by Scion Image software.
Protocol detail
Oxidative Stress Signaling in Muscle Atrophy
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18s rrna
4 hydroxynonenal
Antibodies
Cell cultures
Cytochrome c
Cytochrome oxidase
Gapdh
Hybridoma
Immunoblots
Malondialdehyde
Mnsod
Mrnas
Murf1
Muscles plantaris
Peroxisome proliferator activated receptor γ coactivator 1
Pgc 1α
Primers
Proteins
Real time pcr
Corresponding Organization :
Other organizations : University of Virginia
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Variable analysis
independent variables
- None explicitly mentioned
- MRNA levels of MAFbx/Atrogin-1, MuRF1, and Gapdh
- Protein levels of CuZnSOD, MnSOD, EcSOD, PGC-1α, COX IV, Cyt C, MDA, 4-HNE
- Oxidized protein levels (carbonylated proteins)
- Plantaris muscles
- 18S rRNA as reference gene for real-time PCR
- Validated antibodies and band identification for immunoblot analysis
- None explicitly mentioned
- None explicitly mentioned
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