The degradation rate of DLK protein was analyzed by quantitative immunoblotting after cycloheximide chase assay. Cycloheximide treatments were performed by adding cycloheximide (0.1 g/l; Sigma) without or together with ApoE3 (10 µg/ml) to cultured human neurons on MEFs at D10. At the indicated time points, neurons were lysed and immunoblotted for DLK protein levels. The data of DLK relative intensity at 0h, 2h, 4h and 6h, after normalization to loading control Tuj1, were fit to an exponential decay function (first-order kinetics, A=A0ekt) to estimate the degradation rate constant (k, hour−1). The half-life time (t1/2) is further calculated from the constant (t1/2 = ln(2)/k) to determine the time required for 50% degradation of DLK protein without or with ApoE3.
