We confirmed the presence of DLG4 and KCNMA1 in our brain samples performing a quantitative fluorescent immunoblotting. By using a combination of a total protein staining and an immunostaining on western blot, it is possible to correct for differences in total protein loading. Fluorescent stains allow for peak detection and quantification. For both DLG4 and KCNMA1, presence of the protein in our samples could be established, but no expression differences were detected between disease stages. This in part explains why these proteins were not picked up in our 2D-DIGE analyses. Even though they are not differentially expressed, their presence in the centre of the IPA networks suggests a role for DLG4 and KCNMA1 as central regulators in the molecular mechanisms of disease progression. KCNMA1 is a calcium-activated potassium channel with a direct connection to CAPG, a protein involved in actin-based cell motility and thus important for macrophage functions such as migration and myelin phagocytosis, Nilotinib (monohydrochloride monohydrate)processes known to be highly involved in MS and EAE pathology. We demonstrated that specific blocking of KCNMA1 using paxillin significantly reduced myelin phagocytosis by LPS activated macrophages. This nicely illustrates that the central regulators reported here indeed have a functional role in the disease process. We identified proteins present in 92 differential spots of the inflamed brain of EAE animals by means of a comparative 2DDIGE proteomics analysis. Changes in the abundance of these 92 spots can discriminate between early and late disease stages by PCA of their values in the sample spotmaps. Seventy-five unique proteins were identified in these 92 differential spots by means of mass spectrometry. Some of these proteins represent known disease processes such as BBB disruption, astrocyte activation and macrophage infiltration. Others are not yet linked to MS, and warrant further research. An in-depth network evaluation was performed for all 75 unique proteins. Four central network-nodes were suggested by IPA. DLG4 and APP are nodes from the IPA networks of our data, while KCNMA1 and AGT are MS-related proteins in the IPA knowledge base,GSK 650394 with a strong relationship to our data. APP is an integral membrane protein that is concentrated at neuronal synapses. It can be synthesized by microglia, not only in response to direct nerve injury but also in immune-mediated disease such as EAE. A role for APP in immune and repair mechanisms of the CNS is suggested. A second network is build around AGT, a protein produced by astrocytes in the brain. It is part of the renin-angiotensin system that affects the immune response in general and the neuroinflammatory processes in the context of EAE. TP53, a well-known tumor suppressor that responds to cellular stress and can induce apoptosis and changes in metabolism is also present as a regulator in this network. TP53 was recently described to be an important ‘network-hub’ that interacts with a lot of genes associated with MS, indicating a role for this protein in the disease, namely the expansion of autoimmune cell clones. Sixty-four percent of our data are connected to DLG4 and/or KCNMA1, which highlights their possible role in EAE/ MS. DLG4 is a membrane-associated protein implicated in the clustering of receptors, ion channels and associated signaling molecules in the post-synaptic membrane. It is a key player in neuronal signaling. KCNMA1 is an IPA MS-related protein involved in neurotransmitter release. The channel activity increases during hypoxia and decreases in response to reactive oxygen species. Mitochondrial ion channels play an important role in cellular events such as apoptosis, exocytosis and synaptic transmission and are believed to contribute to cytoprotection.