The lysate was then separated by centrifu gation at 12000 g at 4 C for 15 minutes. The supernatant was collected and the protein concentration was inhibitor Rucaparib measured using a bicinchoninic acid protein assay, Inhibitors,Modulators,Libraries 35 ug samples were loaded into 8% SDS polya crylamide gels. Proteins were then transferred to polyviny lidene difluoride membranes using a 100 V current for 1. 5 hours. The blots were then first washed with Tris buffered saline and Tween, followed by blocking in 5% non fat milk TBS T overnight at 4 C. Antibodies recognizing NF B, TANK binding kinase 1, I B kinase ��, and GAP43 were made up in a solution of in 5% milk in TBS T, and used overnight at 4 C, followed by three washes with TBS T and incubation with horseradish peroxidase conjugated anti rabbit, anti sheep or anti rat IgG secondary antibodies in TBS T for 1.

5 hours at 25 C. The blot was developed with DAB and a commercial chemiluminescent Inhibitors,Modulators,Libraries detection system. Tissue collection and cytokine measurement Real time reverse transcriptase PCR analysis To analyze the mRNA expression of cytokines, total RNA extraction and real time PCR were performed as previously reported, with minor modifications. Total RNA was extracted with 800 ul of the RNA lysis buffer supplied with the kit. RNA was reverse transcribed in accordance with the manufacturers instructions. First strand cDNAs were amplified using a real time PCR thermal cycler. Quantificative real time PCR was performed with Taq polymerase in accordance with the manufacturers instructions. Primers for IFN b, b actin, TNF a, induci ble NO synthase, IL 1b, IL 6, and IL 17 are shown in Table 1.

For relative comparison of each gene, we analyzed the cycle of threshold value of real time PCR data using the Ct method, in accordance with the companys instructions. ELISA analysis Microglial cells were collected at 12, 24, and 36 hours after stimulation of injured RGCs. The Inhibitors,Modulators,Libraries cells were rinsed twice with PBS, and then lysed with a protease inhibitor Inhibitors,Modulators,Libraries cocktail, and frozen at 80 C until analysis. For protein isolation, the samples were milled and separated by centrifugation Inhibitors,Modulators,Libraries at 10, 621 �� g at 4 C for 10 minutes. The supernatant was carefully pipetted into a fresh 1. 5 ml EP Eppendorf tube, and the protein concentration was evaluated by protein assay. For TNF a, IFN b, IL 1b IL 6, and IL 17 detection, a mouse ELISA kit was used, in accordance with the manufacturers instructions.

Briefly, the plate was incu bated with 100 ul of each sample or standard protein, in duplicate. After incubation and subsequent washing, horseradish peroxidase conjugated streptavidin at 400 ng ml detection antibody was added, followed by wash ing and incubation with the substrate solution provided with the kit to produce a color reaction, which was stopped by selleck inhibitor addition of stop solution. The absorbance was read at 450 nm in a microplate reader.