Agarose conjugate was washed twice with washing buffer, centrifuged for 10 sec at 12,000 �� g at room temperature, and then resuspended in washing buf fer. Agarose conjugate was added to 10 ul of anti STAT1 antibody, incubated for 60 min at room temperature with gentle mixing, and then centrifuged at 3,000 �� g for 2 min at 4 C. Samples were washed with 1 ml washing buffer, useful site and centrifuged at 3,000 �� g for 2 min at 4 C, this step was repeated at least twice. Co cultured cell lysates were added to agarose conjugate bound antibody, and incubated overnight at 4 C with gentle mixing. Immunoprecipitated complexes were washed with washing buffer, and centrifuged Inhibitors,Modulators,Libraries at 3,000 �� g for 2 min at 4 C. Pellets were washed with 1 ml washing buffer, and centrifuged at 3,000 �� g for 2 min at 4 C.

This step was repeated at least three times. The pellet was resuspended in 25 100 ul Laemmli sample Inhibitors,Modulators,Libraries buffer. Samples were heated at 95 C for 5 min, centrifuged, and the supernatants were collected. Samples were run on SDS PAGE, transferred to nitrocellulose, and immunoblotting Inhibitors,Modulators,Libraries was performed. Induction of EAE Female mice were purchased from Samtako BioKorea and maintained in specific pathogen free conditions before sacrifice. All mice were housed in accordance with guidelines from the Association for Assessment and Accreditation of Laboratory Animal Care, and all protocols were approved by the Institutional Review Board and conducted in the Laboratory Animal Research Center of Sungkyunkwan University. The EAE model was induced by a method described previously.

Mice Inhibitors,Modulators,Libraries were divided into five groups, control, mice injected with CFA alone, EAE, mice received a subcutaneous injection of 150 ug myelin oligodendrocyte glycoprotein peptide 35 55 in 100 ul PBS mixed with 100 ul of CFA, three treated groups, mice pretreated by intraperitoneal injection of anti CD40 antibody, 8 oxo dG, and a combina tion of both for 5 days after MOG injection, respectively. After MOG injection, each animal received an i. p. injec tion of 200 ng pertussis toxin in 200 ul PBS. The mice were weighed and scored daily in a Inhibitors,Modulators,Libraries blinded fashion by two examiners according to the following scale, score 0, no disease, score 1, loss of weight and tail weakness, score 2, weakness in hind limb, score 3, complete hind limb paralysis, score 4, hind limb paralysis with forelimb weakness or paralysis, and score 5, moribund or deceased.

The concentration of anti CD40 antibody and 8 oxo dG was injected the same amount used in our previous experiments. Thirty two days after starting injection, the EAE score was about 3. 8 0. 21, and brains were isolated, and inflammatory cells infiltrated into brain tissues were determined using hematoxilin and eosin. In general, EAE score reached peak on selleckbio day 21 25, but our EAE score reached peak on day 31 32 despite the same method used in other laboratories. This difference may be due to environmental factors.