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The use of SUnSET as a non‐radioactive method for measuring rates of protein synthesis in whole skeletal muscles and in single fibers
Author(s) -
Goodman Craig Andrew,
Mabrey Danielle M.,
Frey John W.,
Lincoln Hannah C.,
Miu Man Hing,
Schmidt Enrico K.,
Pierre Philippe,
Hornberger Troy A.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.989.25
Subject(s) - sunset , puromycin , in vivo , chemistry , immunohistochemistry , western blot , microbiology and biotechnology , andrology , biology , biochemistry , medicine , protein biosynthesis , optics , immunology , physics , gene
SUnSET (SUrface SEnsing of Translation) involves the immunological detection of puromycin in nascent peptides as a means for quantifying rates of protein synthesis (PS). To validate SUnSET against an established technique, plantaris muscles were subjected to synergist ablation (SA) or sham surgery. After 7d, muscles were incubated ex vivo with a flooding dose of 3 H‐Phenylalanine ( 3 H‐P) or with puromycin. According to the established 3 H‐P method, SA induced a 3.4 fold increase in PS while a western blot (WB) version of SUnSET revealed a 3.6 fold increase, indicating that both techniques produce quantitatively similar results. Furthermore, SUnSET revealed that SA induces a 2.9 fold increase in PS when measurements were performed in vivo . SUnSET was also used to demonstrate that food deprivation induces a ~70% decrease in PS in vivo when measured with WB analysis and similar results were obtained when an immunohistochemical (IHC) version of SUnSET was applied to cross‐sections from control and food deprived samples. Finally, the IHC version of SUnSET was applied to muscles electroporated with constitutively active Akt (CA‐AKT) and it was determined that PS rates were 80% higher in CA‐AKT transfected fibers when compared to non‐transfected fibers in the same section. In conclusion, SUnSET is a simple and inexpensive alternative for measuring PS and allows for measurements to be performed at the single fiber level.