Kristiansen et al. BMC Genomics 2011, 12:551 http://www.biomedcentral.com/1471-2164/12/551 Page 10 of 17 Figure 6 The proteins encoded by the 5 selected genes increase in level after NGF withdrawal. A, Immunoblotting analysis of Trib3, Ddit3, Txnip, Ndrg1 and Mxi1 protein levels using extracts prepared from sympathetic neurons cultured in the presence or absence of NGF ± CEP- 11004 for 16 hours. For all 5 genes, protein levels were significantly increased at 16 hours after NGF withdrawal. Representative images are shown. c-Jun is shown as a positive control. ERK levels are shown as a loading control. The blots were scanned on a densitometer to quantitate the levels of each protein and were normalised to the ERK loading control. The ratio of the level of each protein in the presence of NGF (+NGF) to the level of each protein after NGF withdrawal (-NGF) or in the presence of CEP-11004 was then calculated. B, Analysis of Trib3, Ddit3, Txnip, Ndrg1 and Mxi1 protein levels and subcellular localisation by immunocytochemistry. Sympathetic neurons were treated as indicated and fixed at 0, 2, 4, 8, 12, or 16 hours after NGF withdrawal ± CEP-11004. The cells were then stained with the appropriate primary antibody followed by a FITC-conjugated secondary antibody, and Hoechst dye to label the nuclear DNA. Images were collected using the same exposure time for each timepoint and the coverslips in each gene group were analysed in parallel. The scale bar represents 20 μm.