Fig 1: The MEK-ERK pathway negatively regulates bim mRNA expression in sympathetic neurons. (A) Effect of U0126 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 U0126 at 5, 10, 20 or 40 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 U0126 or LY294002 on Bim protein levels in sympathetic neurons. Immunoblots were performed with whole cell extracts from sympathetic neurons maintained in the presence of NGF, withdrawn from NGF for 16 hours, or treated with 50 µM LY294002, or with 10 µM U0126, for 16 hours in the presence of NGF. Antibodies to detect Bim (Chemicon AB17003) and a-Tubulin (AbD Serotec MCA77G) were used. (C) U0126 does not alter Akt phosphorylation in sympathetic neurons. Immunoblots were performed with whole cell extracts from sympathetic neurons maintained in the presence of NGF, treated with 10 µM U0126 for 16 hours in the presence of NGF, or withdrawn from NGF for 16 hours. Antibodies to detect total Akt (Cell Signaling #9272), phospho-Akt (Cell Signaling #9271) and total ERK1/2 (Cell Signaling #9102) were used. (D) U0126 does not alter c-Jun phosphorylation in sympathetic neurons. Cells were treated as in (C) and antibodies to detect total c-Jun (BD Biosciences #610327), phospho-c-Jun (Santa Cruz sc-822) and total ERK1/2 (Cell Signaling #9102) were used. (E) Total RNA was prepared from sympathetic neurons treated as in (B) and bim mRNA levels were analysed by q-PCR relative to the level of the mRNA encoded by the house-keeping gene Hprt1. The data is presented as the mean ± S.E., n = 3. Endogenous bim mRNA levels increased significantly following NGF withdrawal and treatment with LY294002, respectively (p = 0.015 and p = 0.014). Treatment with U0126 also induced a significant increase in the level of bim mRNA (p = 0.0003).