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.
Department of Cell Biology and Neuroscience, University of South Alabama College of Medicine, MSB2042, Mobile, AL 36688, USA.
J
J
Sangerman
S R
SR
Goodman
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