6 to 2. 5 mm. A single optical slice was continuously Seliciclib CDK2 Inhibitors,Modulators,Libraries but sequentially scanned for DiIc18, RH237, and Fluo 4. Images were collected and processed using LaserSharp, LaserPix, and LaserVox. Media products Media products DMEM, DMEMF12, FCS, HI FCS, gentamycin sulfate, and L glutamine were obtained from Biological Industries. Statistical analysis Mann Whitney and one and two way ANOVA analyses were carried out as indicated in the figure legends. Results Syk is activated during phagocytosis of degenerated myelin Phagocytosis of myelin in the absence of anti myelin Abs is mediated jointly by CR3 and Inhibitors,Modulators,Libraries SRA, albeit two to four fold more by CR3 than SRA.

Taken that Syk is activated by phosphorylation during CR3 mediated Syk depended functions, we tested whether Syk is also phosphorylated during myelin phagocytosis in wild type BalbC microglia and microglia infected Inhibitors,Modulators,Libraries with the non target luciferase shRNA that were used in subsequent experiments as control for micro glia that were infected with Syk shRNA. Levels of p Syk increased substantially for the en tire 30 min period of phagocytosis tested, indicating that Syk was continuously activated during prolonged phago cytosis. Syk down regulates CR3 mediated myelin phagocytosis We examined next how Syk regulates myelin phagocytosis by analyzing how two distinct inhibitors of Syk affected phagocytosis. The inhibitor SYK inhibitor augmented phagocytosis of C3bi opsonized and unopsonized myelin, which is mediated jointly by CR3 and SRA in wild type BalbC microglia and by CR3 without SRA in BalbC SRA microglia.

SYK inhibitor augmented phagocyt osis of C3bi opsonized and unopsonized myelin by CR3 and SRA combined in wild type C57BL microglia but pro duced inhibition of phagocytosis by SRA without CR3 in C57BL CR3 microglia only at 1 uM. Finally, SYK inhibitor augmented Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries phagocytosis of C3bi opsonized and unopsonized myelin in wild type BalbC and C57BL macrophages. Taken together, SYK inhibitor augmented phagocytosis of C3bi opsonized and unopso nized myelin by CR3, but not by SRA. In contrast to aug mentation by SYK inhibitor, phagocytosis of C3bi opsonized myelin was reduced in wild type BalbC and C57BL microglia by piceatannol, which was frequently used by others. The opposing effects that the two inhibitors of Syk produced did not allow us to determine whether Syk activated or downregulated myelin phagocytosis.

We addressed this issue further by examining how phagocyt osis is affected after reducing Syk expression in phago cytes. Syk was knocked down by lentiviral infection with Syk shRNA in wild type BalbC microglia. Consequently, phagocytosis of C3bi opsonized and unopsonized myelin was augmented, suggesting find FAQ that normally Syk downregulates this phagocytosis. Thus, augmentation in Syk KD micro glia corresponded to augmentation by SYK inhibitor and not to inhibition by piceatannol, indicating that SYK in hibitor and not piceatannol is Syk specific in our assay system.