Fig. 3AExtracellular signal-regulated kinase (ERK) pathway regulates the neurogenic differentiation 1 (NeuroD1) protein stability via ubiquitination. (A) Western blot analysis of NeuroD1 protein in the absence and presence of PD 98059, a mitogen-activated protein kinase (MEK) inhibitor. Cycloheximide (CHX) was added 30 min prior to the addition of PD 98059. (B) The NeuroD1 protein intensity shown in (a) was presented with respect to the value at t=0 (n=3). (C) Western blot analysis of Myc-tagged mutants of NeuroD1 protein at ERK phosphorylation sites with and without N-acetyl-Leu-Leu-norleucinal (ALLN), a proteasome inhibitor. CHX was added 30 min prior to adding ALLN. (D and E) NeuroD1 protein intensity shown in (c) was presented with respect to the value at t=0 (n=3). Note that the S274A mutant exhibited the highest half-life and ALLN increased the half-lives of the wild type (WT) as well as all mutants. (F) Western blot analysis showing the ubiquitination of NeuroD1 WT in the presence of ALLN. PD 98059 partially attenuated ubiquitination. (G) Myc-tagged mutants of S259A, S266A, S274A, or Triple-A were analyzed for ubiquitination. The experiments were repeated 3 times and the most representative results are shown in , C, F and G. Note that S274A abolished ubiquitination, whereas S266A showed a lower level of ubiquitination compared to the WT.
© Exp Neurobiol
