The initial discovery phase combined iTRAQ labelling with off-line twodimensional liquid chromatography tandem MS

ffer was added. Cells were incubated for 10 minutes on ice, then buy Pomalidomide scraped into an eppendorf tube. The lysate was spun at 4 degrees Celsius in a table top microcentrifuge for 10 minutes at 16,000 g to pellet cell debris. The protein concentration of the resulting supernatant was quantified using a BCA assay. Western Blots For all western blots, 20 micrograms of total protein was loaded per sample. For all western blots except MMP-2, lysates were run on 9% acrylamide running gels with a 4% acrylamide stacking gel. Lysates blotted for MMP-2 were run on precast 420% gradient gels. Gels were run at 185 volts constant. Protein was then transferred to 0.45 mm nitrocellulose using a ThermoFisher electrotransfer apparatus at 0.4 amps constant. After transfer, the nitrocellulose membrane was blocked for 1 hour at room temperature in 5% dry milk in 0.05% Tween20-TBS or in 0.05% Tween20-TBS. Primary antibodies were diluted in blocking solution and incubated overnight at 4 degrees Celsius. The blots were washed 3 times in 0.05% Tween20-TBS, and secondary antibodies were diluted in 0.05% Tween20-TBS and incubated with the blot for 1 hour at room temperature. The blots were washed with 0.05% Tween20-TBS three times, then developed using West Pico reagents. Retroviral Transduction The stable cell lines sh5, sh6, 2C-ALC, and sh5rxd were created by retroviral transduction. The appropriate constructs were cotransfected, along with pCMV-VSV-G, into the GP2-293 packaging cell line. Conditioned media was harvested from the GP2-293 cells, filtered, supplemented with 4 mg/ml polybrene, and added to the appropriate parental cell line. The following day, the media was removed from the parental cell line, and conditioned media from the GP2-293 cells was added again. This process was repeated a total of three times. After the final day of incubation in conditioned media, the parental cells were grown for 24 hours in standard growth medium, and then appropriate selection agents were added. Gelatinase Assay Gap Closure Assay/time-lapse Imaging Confluent cultures were grown in 6 cm dishes. A 10 ml pipet tip was used to inscribe a ��wound gap��in the confluent layer of cells. Cells were washed twice with 16PBS, then RPMI-1640 media containing 10% FBS and 25 mM HEPES was added. The dishes were covered and placed on a custom stage heater that maintained media temperature at,37 degrees Celsius. To analyze cell motility, phase contrast time-lapse microscopy was done using a QICAM camera and 106 objective on a Leica DM-IRB microscope. Images were collected every 10 minutes for 8 hours, or until cells closed the wound gap. Analysis of cell migration was conducted by using QImagePro. The initial width of the gap was measured at three different places along the length of the wound, and the time until the cells closed the gap was recorded for each. The three speeds were calculated and averaged together to yield a value for that trial. Each reported speed represents at least three independent trials. Cells were grown to confluency in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22201297 a 10 cm dish. Growth medium was removed and replaced with 5 ml of serum-free medium for 36 hours. The media were harvested, filtered through a 0.45 micron filter to remove cells and debris, and mixed 1:1 with non-reducing sample buffer. Samples were treated as in, and 10 ml loaded onto an SDS-PAGE gel that included 0.1% gelatin. Invasion Assay The CytoSelectTM 24-well Invasion Assay, Basement Membrane, Colorimetric format was used according to manufac

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