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Intracellular protein turnover

BC Benjamin Caballero
MB Mathieu Bourdenx
EL Enrique Luengo
AD Antonio Diaz
PS Peter Dongmin Sohn
XC Xu Chen
CW Chao Wang
YJ Yves R. Juste
SW Susanne Wegmann
BP Bindi Patel
ZY Zapporah T. Young
SK Szu Yu Kuo
JR Jose Antonio Rodriguez-Navarro
HS Hao Shao
ML Manuela G. Lopez
CK Celeste M. Karch
AG Alison M. Goate
JG Jason E. Gestwicki
BH Bradley T. Hyman
LG Li Gan
AC Ana Maria Cuervo
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To measure the degradation of long-lived proteins, confluent cells were labeled with 3H-leucine (2 μCi/ml) for 48 h at 37 °C, transfected with the indicated plasmids and then extensively washed and maintained in complete (10% FBS) or serum-deprived media containing an excess of unlabeled leucine (2.8 mM) to prevent reutilization of radiolabeled leucine47. Aliquots of the media taken at different times were precipitated with TCA and proteolysis was measured as the percentage of the initial acid-insoluble radioactivity (protein) transformed into acid-soluble radioactivity (amino acids and small peptides) at the end of the incubation. Total radioactivity incorporated into cellular proteins was determined as the amount of acid-precipitable radioactivity in labeled cells immediately after washing.

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