logy. Monoclonal antibodies against v3 integrin and 51 were from Chemicon. Anti uPAR mAb was from American Diagnostica Inc. Rabbit polyclonal anti uPAR antibody was a gift kindly provided Tosedostat Androgen receptor inhibitor by Drs. Andrew Mazar and Graham Parry. Vascular endothelial growth factor and basic fibroblast growth factor was obtained from Invitrogen Corporation. All other reagents were purchased from Sigma Chemical unless otherwise specified. Preparation of recombinant D5 of HK Glutathione S transferase and recombinant GST D5 were prepared as previously described. Briefly, GST was removed from GST D5 by digestion with thrombin, which was inactivated with d phenylalanyl l prolyl l arginine chloromethyl ketone. Free GST was removed with Glutathione Sepharose 4 Fast Flow column. Residual thrombin and PPACK were removed with Amicon Centriprep YM 30.
Using YM 10, D5 solution was exchanged into 50 mM HEPES, 150 mM NaCl, pH 7.5 buffer. Endotoxin levels in the preparations were determined with the Crenolanib 670220-88-9 chromogenic limulus amebocyte lysate assay by use of an endotoxin testing kit. Endotoxin level in D5 was below detectable limits. D5 was visualized on 20% SDS PAGE and detected by Western blotting as a single band. Cell Culture DU145, a prostate cancer cell line, was purchased from ATCC. DU 145 cell line was maintained in Dulbecco,s Modified Eagle,s Medium containing 10% fetal bovine serum, 2mmol/L glutamine, 100 units/mL penicillin, and 100 g/mL streptomycin and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37. Zn were added to the culture mix whenever HKa and D5 were involved, as Zn is required for HKa and D5 binding to tumor cells.
Cell Migration Assay Cell migration was assessed in 48 well Boyden chambers. The under side of membrane of the upper chamber was coated with a collagen mixture and DU145 cells in DMEM were seeded on the upper chamber. DMEM contained bFGF was added to the bottom chamber. Tumor cells were allowed to migrate for 6 hrs. Then, the cells that remained in the upper chamber were removed using a cotton swab. The cells that migrated to other side of membrane of the upper chamber were fixed with 4% paraformaldehyde and stained with 1% toluidine blue. We counted cells in 5 fields per well that essentially covered 80% of the well surface. The average number of cells from each of the triplicates represents the average number of cells that migrated in that treatment group.
Each experiment had triplicate wells for every treatment group and we repeated each experiment three times. The mean of all results from controls was considered as 100%. Cell Invasion Assay Cell invasiveness was determined by the ability to transmigrate through a layer of Matrigel in a Transwell chamber. Briefly, the 1:1 mixture of matrigel and DMEM was loaded on the top chamber of Transwell units. DU145 cells were loaded on the top of matrigel. The medium 10% FBS Zn was added to the bottom chamber of Transwell units. Twenty four hrs later, cells were fixed by formaldehyde and stained by 1% Liu et al. Page 3 Oncogene. Author manuscript, available in PMC 2010 April 28. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript toluidine blue. The cells that remained in the upper chamber were removed using a cotton swab. Cells which migrated to the underside of a membrane were counted as described in Cell Migration Assay. Cell Lysate Preparation, Immunoprecipitation