Immunoblot analysis The effects of amuvatinib on HGF dependent s

Immunoblot analysis The effects of amuvatinib on HGF dependent signaling were assessed in U266 cells that had been serum starved for 24 h in RPMI 1640 containing 0. 1% FBS. for the last 16 h of starvation the cells were treated with various concentrations of amuvatinib or DMSO. They were then treated with 50 ng/ml HGF for 15 min to stimulate MET. Amuvatinib mediated induction of PARP cleavage was performed on U266 cells cultured in full serum as well as under low serum conditions. Protein lysates and immunoblots were prepared as previously described. Experiments were performed in triplicates, and bands were quantified by using an Odyssey Infrared Imaging System. Primary antibodies were mouse monoclo nal antibodies to MET clone 3D4 . GSK 3B clone 7/GSK 3b, PARP clone C2 10, cleaved PARP Asp 214 clone F21 852, AKT clone 9Q7 .

GAPDH clone 6C6 . phospho ERK1/2 clone E10 . B actin clone AC 15 . rabbit monoclonal antibodies to phospho GSK 3B clone 5B3 . rabbit polyclonal antibodies to phospho MET . ERK1/2, and phospho AKT. Flow cytometry Intracellular protein expression in U266 cells was mea sured using BD Cytofix/Cytoperm Fixation/Perme abilization Kit. Primary antibodies used were anti phospho HGF R/c MET , MET sc 10, phospho AKT, AKT antibody . and caspase 9. Secondary antibody was a fluorescein isothiocyanate conjugated Affinipure goat anti rabbit. Cell cycle analysis and annexin V/propidium iodide stain ing were performed, respectively, as described. All flow cytometry analysis was performed using a Becton Dickinson FACSCalibur flow cytometer.

Statistical significance of changes was assessed by paired t test analysis using Prism software. Enzyme linked immuno sorbent assay for HGF levels HGF levels in primary patient plasma were determined using the Human HGF Immunoassay Kit as per the manufacturers protocol. The absorbance of this horseradish peroxidase based assay was measured at 450 nm. Each sample was assayed in triplicate. Background Farnesyl transferase and Geranylgeranyl trans ferase I are heterodimeric enzymes that cat alyze the transfer of C 15 or C 20 lipid moieties, respectively, to the C terminal cysteine of proteins hav ing CAAX motifs at their C terminus, the last amino acid discriminating among the two enzyme substrates. The observation that Ras oncoproteins require far nesylation for membrane binding and malignant activity AV-951 led to the development of drugs targeting FTase.

As FTase structure and function has been conserved throughout evolution, the first farnesyl transferase inhi bitor, Manumycin A, was selected using a yeast based screening system. Over the past decade, improved chemically synthesized FTase and GGTase I inhibitors were tested in preclinical models. Surprisingly, they were active on a wide range of tumors independently of their Ras oncogenic status.

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