The MEK-ERK pathway negatively regulates bim expression via regulatory elements outside of the bim promoter, exon 1 and intron 1. Sympathetic neurons were microinjected with the bim-LUC construct (10 ng/μl) along with pRL-TK (5 ng/μl) to control for injection efficiency. The cells were either maintained in medium containing NGF, withdrawn from NGF, treated with LY294002 at 50 μM, or with U0126 at 10 μM, in the presence of NGF. After 16 hours, 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 is activated significantly following NGF withdrawal or after treatment with LY294002, respectively (p = 0.003 and p = 0.001). However, treatment with U0126 does not activate the bim-LUC construct.