Measurement of the synthesis, turnover, and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons.
We describe a method that has allowed us to measure the synthesis, turnover and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. For these studies, rat hippocampal cultures containing 74.5-83.0% neurons were established. B-27 (Gibco) supplement has been used to obtain an excellent long-term viability (up to 5 weeks) of hippocampal neurons in culture. For the synthesis, turnover, and assembly experiments the neurons were labeled with [35S]methionine, and chased with 10-fold excess of cold methionine for the turnover experiments. The cells were then lysed and immunoprecipitated with alpha, beta-erythroid, alpha, and beta-nonerythroid spectrin antibodies. Immunoprecipitated [35S]methionine-labeled spectrins of hippocampal neurons grown in vitro produced bands in 5% polyacrylamide minigels strong enough to be detected by the high sensitivity screens of a phosphorimager to generate graphs from which the synthesis or half-lives of alpha, beta-erythroid, alpha, and beta-nonerythroid spectrins were calculated. This method can be used to study the role of calpain, caspase-3, and the ubiquitin-proteasome system on the synthesis and turnover of erythroid and nonerythroid spectrins in resting and depolarized rat hippocampal neurons in culture.
Sangerman
J
J
Department of Cell Biology and Neuroscience, University of South Alabama College of Medicine, MSB2042, Mobile, AL 36688, USA.
Goodman
S R
SR
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D005260:Female; D006624:Hippocampus / Q000166:cytology; D008297:Male; D008715:Methionine / Q000493:pharmacokinetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D013049:Spectrin / Q000096:biosynthesis* / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D013462:Sulfur Radioisotopes; D013049:Spectrin; D008715:Methionine