The MEK1/2-ERK1/2 pathway negatively regulates bim mRNA expression in sympathetic neurons. (A) Effect of PD184352 on phosphorylation of ERK1/2 in sympathetic neurons. Immunoblots were performed with whole cell extracts from sympathetic neurons maintained in the presence of NGF and left untreated or treated with PD184352 at 0.1, 1, 2 or 10 μM for 16 hours. Antibodies to detect total ERK1/2 (Cell Signaling #9102) and phospho-ERK1/2 (Cell Signaling #9101) were used. (B) Effect of PD184352 on phosphorylation of ERK5 in sympathetic neurons. Cells were treated as in (A), but including a set of extracts prepared from cells withdrawn from NGF for 16 hours, and antibodies to detect total ERK5 (Cell Signaling #3372S) and phospho-ERK5 (Santa Cruz sc16564) were used. (C) Q-PCR data re-plotted from Figure 1E, but including the final data group where neurons were treated with PD184352 at 2 μM for 16 hours in the presence of NGF. The data is presented as the mean ± S.E., n = 3. Endogenous bim mRNA levels increased significantly following NGF withdrawal (p = 0.015), treatment with LY294002 or U0126, respectively (p = 0.014 and p = 0.0003). Treatment with PD184352 also induced a significant increase in the level of bim mRNA (p = 0.05). (D) Sympathetic neurons were microinjected with bim-LUC at 10 ng/μl or bim-LUC+3'UTR at 14 ng/μl, along with pRL-TK (5 ng/μl). The cells were maintained in medium containing NGF and either left untreated (-PD184352) or treated with PD184352 at 2 μM (+PD184352) for 16 hours, after which time luciferase activity was measured. Relative luciferase activity was determined by normalisation of Firefly luciferase activity to Renilla luciferase activity (pRL-TK refers to Renilla luciferase under the control of the thymidine kinase promoter). The data is presented as the mean ± S.E., n = 4. Bim-LUC+3'UTR is activated significantly following inhibition of the MEK1/2-ERK1/2 pathway by treatment with PD184352 (p = 0.005). Bim-LUC is not activated significantly following treatment with PD184352. (E) The basal levels of bim-LUC and bim-LUC+3'UTR were normalised to 1 and the induction factors of the two constructs were compared. Addition of the bim 3' UTR significantly increases the induction of bim-LUC upon inhibition of the MEK1/2-ERK1/2 pathway (p = 0.009).