with 10 KK Calf serum and grown in NVP-LAQ824 culture contains kept in a humidified incubator with CO2 Lt consists of 10 to 37. NVP AEW541 was a gift from Novartis Pharma AG WAW. NDGA was a gift from Insmed Corporation. Coffee S ure, 5,8,11 Eicosatriy??no only, and cinnamyl 3.4 dihydroxy cyanocinnamate were purchased from Biomol International. NVP AEW541, S Acid coffee, ETI, CDC and NDGA were immediately before each experiment was in DMSO gel L 1000 St. IGF-I purchased from GroPep. Anti-IGF-IR ancient body was purchased from Calbiochem chem. Anti-insulin receptor developed in Goldfine’s lab at the University of California at San Francisco. Phosphotyrosine and anti-GAPDH old bodies were purchased from Santa Cruz Biotechnology. Anti-Akt, phospho Akt antique Body ERK 2 1 phosphorylated ERK1 fight 2 and cleaved caspase 3 anti Older instances of Cell Signaling Technology were acquired. Horseradish peroxidase conjugated goat anti-rabbit IgG was purchased from Zymed Laboratories.
CyQUANT was purchased from Molecular Probes. Propidium iodide were purchased from Sigma. IR and IGF InsR phosphorylation ELISA SH SY5Y and SHEP cells were grown to confluence in DMEM 80 K Calf serum culture VX-745 grown 10 K, followed by serum starvation Final 4 h Cultures were then treated with DMSO or 60 M NDGA and incubated for 1 h . Some cultures were then treated with 1 nM of IGF-I for 10 min. The medium was removed, three cultures rinsed in cold PBS and lysis buffer was added. The cultures were rocked in lysis buffer at 4 for 1 h. Ninety-six wells were rpern with antique plates or organization MA-20 IR 3 antique in 50 mM NaHCO 3, pH 9.0 for 2 h are at room temperature. The plates were 3 in Tris Tween 0.1 Salzl L Purged solution and blocked with Superblock 30 min at room temperature. Each well of the ELISA plate were again U follows 30 g of protein from cell lysates by incubating cultures for 24 h at 4.
The plates were washed with TBST 5 and HRP-conjugated anti-phosphotyrosine old K Body was washed for 2 h at room temperature, rinsed added. The plates were again washed in TBST, and 5 TMB was added until the blue color was sufficiently developed. Absorbance at 451 nm was measured. Each condition was performed in triplicate, and the experiment was repeated three times. CyQUANT assay for cell growth, the cells were cultured in four 96-well plates for tissue culture in DMEM K K calf serum were plated to 10 with a density of 8000 cells and well-grown for 24 hours. In a series of experiments, serum-free DMEM with 1 bovine serum albumin has been set. IGF-I was added to some samples. In a second series of experiments, the cells were cultured in DMEM serum nor K plane 10 were cultured for a period of the experiment without IGF-I in all the experiments, or DMSO or varying concentrations of NVP AEW541, CDC, acidic coffee, EIT and NDGA added to the cultures at 0 h, the dose range of each compound on their respective IC 50 for the S singer IGF-IR or lipoxygenase base used. Plate medium was removed and the plate was frozen ? This plate 0th s