Tao He, Hong Li, Rong-hui Li, Cheng-Gang Duan, Lin Gan, Juan Li, Jie Song
OBJECTIVE: To develop a new method for analysis of protein phosphorylation modification in cultured neuron. METHODS: Cultured neurons were pre-incubated in DMEM without sodium phosphate for 15 min to deplete the metabolic pools. Neurons were then labeled with [32P] orthophosphate (2.78 x 10(6) Bq/ml) for 1.5 h and stimulated by either insulin (100 nmol/L), EGF (20 nm/L) or saline for 0, 5, 20, 60, 120 min. Reactions were terminated by freezing neurons in liquid nitrogen prior to the solubilizing of them in a lysis buffer containing 8 mol/L urea, 4% CHAPS, 2% Bio-lyte, pH 3-10, 2 mmol/L TBP...
September 2004: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition