have shown that Akt dependent phosphorylation of CavB2, the chaperone of the L

4 Qui nazolinamine, N 7 methoxy 6 was a gift from Astra Zeneca, and cetuximab was kindly provided by Merck KgaA. thymidine and myo inositol were from Amersham Biosciences. Antibodies against phosphory lated AktSer473, total Akt, dually phosphorylated ERKThr202 Tyr204, phospho EGF receptorTyr1173, and phospho Shc Tyr239 240 were obtained from Cell Signal ing Technology. Anti ERK and INNO-406 臨床試験 anti Shc antibodies were obtained from Upstate. EGFR antibody was obtained from Santa Cruz Biotechnology, Inc.. Secondary antibo dies were purchased from Bio Rad Laboratories and Licor Biosciences. All other chemicals were of analytical quality. Stock solu tions of test compounds were prepared in DMSO or 0. 9% NaCl. EGF was dissolved in 4 mM HCl, and TGFa in 4 mM HCl containing 1% bovine serum albumin from Sigma Aldrich.<br><br> Cetuximab was dissolved in phosphate buffered saline. When solutions con taining DMSO were used, the final concentration of DMSO was kept as low as Lapatinib 構造 possible. Cell culture Human colorectal cancer cell lines HCT116 and HT29, and pancreatic adenocarcinoma cell line Panc 1 were obtained from ATCC. The cells were maintained in Dulbeccos modified Eagles medium con taining 1 g l glucose supplemen ted with 10% horse serum, penicillin, streptomycin and 2 mM glutamine. Cells were plated onto Costar plastic culture wells at a density of 50 000 cells cm2 in serum containing medium. The cultures were kept in 95% air 5% CO2 at 37 C. After 24 hours the medium was replaced with serum free medium and the cells were cultured for 24 hours before stimulation with agonists.<br><br> Measurement of DNA synthesis Neurotensin, TPA, and inhibitors of PKC and EGF receptor were added to serum starved HCT116 cells as described in the figure legends, and thymidine was added 12 hours LY2109761 after stimulation. Serum starved HT29 and Panc 1 cells were stimulated for 21 hours with neurotensin and EGF before thymidine was added. The cells were harvested after three hours pulsing with thymidine, and DNA synthesis was measured as the amount of radioactivity incorporated into DNA as previously described. Briefly, medium was removed, and cells were washed twice with 0. 9% NaCl. The cellular material was dissolved with 1. 5 ml of 0. 5 N NaOH for 3 hours at 37 C, collected, mixed with 1. 5 ml H2O, and precipitated with 0. 75 ml 50% trichloroa cetic acid. The acid precipitable material was transferred to glass fiber filters and washed twice with 5.<br><br> 0 ml 5% TCA, followed by liquid scintillation counting of the filters in a Packard Tri Carb liquid scintillation counter. Inositol phosphate accumulation Cells were labelled with inositol, 2. 5 uCi ml for 24 hours in serum free medium. Medium was removed 30 minutes before agonist stimulation and replaced with Krebs Ringer Hepes buffer pH 7. 4, containing 10 mM glucose and 15 mM LiCI. HCT116 cells were stimu lated with neurotensin for 30 minutes, and the reaction was stopped by removing buffer and adding 1 ml ice cold 0. 4 M perchloric acid. Samples were harvested and neutralized with 1. 5 M KOH, 60 mM EDTA, 60 mM Hepes, in the presence of Universal indicator. The neutralized supernatants were applied on columns con taining 1 ml Dowex AG 1 X8 resin, and inositol phosphates were eluted with 10 ml 1 M ammonium formate 0.