1% Tween 20. Primary selleck chem inhibitor antibodies were diluted according to the manufacturers instructions and membranes incu bated overnight at 4 C. After washing, the membranes were incubated with horseradish peroxidase conjugated anti rabbit or anti mouse IgG antibodies, and proteins were visualized using ECL immunoblotting detection systems from Roche Applied Science on a cooled charge coupled Inhibitors,Modulators,Libraries device camera. Densitometrical analysis of the immunoblots Inhibitors,Modulators,Libraries was per formed using advanced image data analyzer soft ware. Apoptosis assay Control and Rictor null MEFs were starved for 24 h, then the extent of apoptosis was determined by quantifi cation of nucleosomes released into the cytoplasma using the Cell Death Detection ELISA Plus kit according to the manufacturers direc tions.
In the separate experiments the level of caspase 3 cleaved fragments was analyzed by immunoblotting. 3 For thymidine incorporation assay subconfluent cell cultures were serum starved in 24 well plates and then incubated for 24 h in the presence Inhibitors,Modulators,Libraries or absence of rapa mycin with PDGF BB in DMEM containing thymi dine. Incorporation of 3H radioactivity into Inhibitors,Modulators,Libraries acid insoluble material was measured by a scintillation counter. The obtained count per minute values in tri plicate was normalized against the positive control of cultures incubated in 10% bovine serum for each experiment. Cell migration assays Cell migration was determined as previously described. In brief, 96 well ChemoTX cell migration microplate filters were coated with 50 ugml fibronectin for 1 h at room temperature.
Control Inhibitors,Modulators,Libraries and Rictor null MEFs, or NIH3T3 cells treated with or without rapa mycin, were serum starved overnight and then trypsinized into single selleckbio cells. The wells of the ChemoTX microplate were filled with DMEM containing the indicated PDGF BB concentrations. The filters were placed in the wells and 50,000 cells were added on top of each filter. The chamber was incubated for 4 h at 37 C, 5% CO2. Cells adhering to the bottom of the filter were fixed by a 3 min incubation in 96% ethanol. The adherent cells were stained with Giemsa and the migration indices were assessed by scanning the filter in a CCD camera. Quan tifications were performed using Aida Image Analyzer software. All experiments were performed in quadru plicate, and single representative data of three separate experimentsSD are shown. Background Enteropathogenic Escherichia coli are an important cause of infantile diarrhea, especially in developing coun tries. EPEC adhere, and cause the local effacement of the microvilli of intestinal epithelial cells, giving raise to so called attaching and effacing lesions. In vitro, EPEC attach to infected cells by forming pedestal like structures enriched in polymerized actin and other host cell proteins.