4 GBR 12909 was added for 60 min at 37 C. In experiments containing 50 nM reserpine, a VMAT inhibitor, a 120 min preincuba tion in uptake buffer preceded the 60 min GBR 12909 pre incubation. GBR 12909 was added to define selective efflux by DAT. In experiments containing kinase inhibitors 10M U0126 or 10M Ly294002 were also added during the 60 min uptake buffer addition. things 10M H89 and 100 nM Ro32 0432 were added to the uptake buffer for 30 min of preincubation. For experiments testing Ca2 involvement, 1M thapsi gargin was added for a 15 min preincubation to empty intracellular Ca2 stores, or cells were incubated for 10 min in 0 Ca2 medium and washed twice in 0 Ca2 medium. For all assays cells were loaded with 3H DA for 10 min prior to Inhibitors,Modulators,Libraries two washes in release buffer.
Release buffer containing treatments, GBR12909, was then added, and extracellular fluid was collected at 9 min to assess3H DA efflux. Inhibitors,Modulators,Libraries Triplicate aliquots were counted in 2 ml Scintiverse II scintillant using a Beckman LS600SE scintillation counter. Specific efflux was defined by averaging the disintegrations per minute due to efflux in the presence of desipramine and GBR 12909, and then subtracting these values from the efflux observed with desipramine alone. We subtracted background from treatment groups and represented the data as 3H DA efflux compared to % of 9 min 10 9 M E2 induced Inhibitors,Modulators,Libraries efflux. Co Immunoprecipitation PC12 cells were collected from five, 150 cm2 Corning tis sue culture flasks by scraping, and then centrifuged at 1500 g, 4 C for 5 min, and resuspended in 2 ml homog enizing buffer.
Cells were then sonicated 15 Inhibitors,Modulators,Libraries times using a pulse probe sonicator, and further processed using a Dounce Inhibitors,Modulators,Libraries homogenizer, on ice, until the majority of cells appeared broken by microscopic examination. The result ing broken cell preparation was then centrifuged at 1500 g at 4 C to remove the nuclear pellet. The supernatant was then centrifuged at 120,000 g at 4 C to obtain the plasma membrane pellet, which was then resuspended in membrane buffer by stirring 8 hours at 4 C and then re pelleted by centrifuga tion for 45 min at 45,000 g, 4 C. The Bradford Bio rad assay was used to determine protein concentration in the supernatant per manufacturers instructions. Protein sam ples were incubated with 40l protein G agarose beads for 10 min at 4 C, then centrifuged using a microfuge for 1 min.
Romidepsin structure The supernatant was incubated overnight at 4 C with 2. 5g DAT antibody. 50l of protein G agarose beads were washed 3 times in phosphate buffered saline and samples containing antibody were incubated with these beads for 4 hours at 4 C on a rotator. Beads were then washed 4 times with PBS for 10 min, each wash. Samples were eluted using 50 mM glycine buffer pH 2. 5, added to SDS sample buffer and heated at 67 C for 10 min, and then electrophoresed on a 7. 5% acrylamide SDS PAGE gel followed by transfer to a nitrocellulose membrane. Blots were blocked using 2. 5% BSA and 2.