Protein was applied to 4 20% Tris SDS Criterion gels, and separated proteins electro www.selleckchem.com/products/SB-203580.html transferred onto Immobilon P PVDF membranes. The membranes were blocked for 30 min. at room temperature in 100 mM Tris buffered saline pH 7. 4 with 0. 1% Tween 20 supplemented with the indicated concentration of non fat dry milk, and incu bated overnight at 4 C with primary antibodies diluted in blocking buffer with milk or bovine serum albumin, as described in Table 1. After wash ing, blots were incubated with horseradish peroxidase conjugated secondary antibodies at the indicated dilu tion for 1 hr at room temperature, and protein bands were visualized by chemiluminescence on X ray film as previously described. Antibodies against phospho specific proteins were applied to freshly trans ferred membranes.

After detection, membranes were stripped with 1 M Tris HCl buffer Inhibitors,Modulators,Libraries containing 2% SDS and 0. 86% 2 mercaptoethanol in Inhibitors,Modulators,Libraries a 50 C hybridization oven for 60 min, and probed with antibo dies against total protein levels as indicated. Equal pro tein loading was confirmed by b actin levels and Coomassie gel staining. Band density was quan tified by Un Scan It software, Inhibitors,Modulators,Libraries and values normalized either to b actin or relevant total protein bands on each PVDF membrane. Drug treatment of cells To selectively block activation of the Erk and Akt signal ing pathways, selective inhibitors of MEK and PI3K were used at 5 uM and 10 uM, respec tively. Drugs were dissolved in DMSO in amber tubes immediately prior to use, and added in SF MEM a to cells cultured alone, with MH S macrophages, with M CM, or with recombinant growth factors for 72 hrs.

The concentration Inhibitors,Modulators,Libraries of DMSO in all experiments never exceeded the vehicle control of 0. 05%. To selectively Inhibitors,Modulators,Libraries block IGF 1R signaling, NVP AEW541 was directly dissolved in to 0. 5% BSA supplemented MEM a media, and added to cell containing wells at a final concentration of 5 uM. Statistical analysis and estimation To estimate the size of the M CM factor responsible for stimulating neoplastic proliferation, we derived a function describing the extent of tumor cell growth in terms of the size of molecules predicted to be con tained in isolated fractions of conditioned media. The percent retention on size exclusion columns vs. protein size on each size m. w. c. o. column was provided by the manufacturer for six recombinant proteins of varying size.

The resulting data set was plotted as per cent retained vs. protein size, and the least complex best fit equation was obtained using non linear regres sion with SigmaPlot 2001 ver 7. 101. The extent selleck screening library that total, unfractionated M CM stimulated LM2 proliferation was normalized to 100%. The extent that each retentate fraction stimulated LM2 growth was similarly calculated to determine the remaining percent of growth stimulating ability after filtration, as compared to unfractionated M CM.