The results sug gested that even though neurotensin acted predominantly by PKC in Panc one cells and by means of EGFR transactiva tion in HT29 cells, it used each these pathways in HCT116 cells. Inside the latter cells neurotensin induced activation of ERK was mediated largely by PKC, while neurotensin induced activation of Akt was independent of PKC but involved transactivation with the EGFR, appar ently by a Ca2 dependent mechanism. Neurotensin induced DNA synthesis was mediated largely by PKC.
Techniques Chemical substances Dulbeccos modified Eagles medium, N piperazine N penicillin and streptomycin were from Gibco Neurotensin, twelve O tetradecanoylphorbol 13 acetate thapsigargin, epidermal development issue and wortmannin selleckchem TW-37 had been obtained from Sigma Aldrich maleimide four 6,seven dimethoxyquinazoline two amino 3 methoxyflavone two four methylpentanoyl Mubritinib L tryptophan methylamide have been from Calbiochem seven Methyl two 9 4H pyrido pyrimidin four one was obtained from Cayman Chemical Transforming development factor a was obtained from Bachem 4 Qui nazolinamine, N seven methoxy six was a present from Astra Zeneca and cetuximab was kindly presented by Merck KgaA thymidine and myo inositol had been from Amersham Biosciences Antibodies towards phosphory lated AktSer473, total Akt, dually phosphorylated ERKThr202 Tyr204, phospho EGF receptorTyr1173, and phospho Shc Tyr239 240 have been obtained from Cell Signal ing Engineering Anti ERK and anti Shc antibodies had been obtained from Upstate EGFR antibody was obtained from Santa Cruz Biotechnology, Inc. Secondary antibo dies had been purchased from Bio Rad Laboratories and Licor Biosciences All other chemicals had been of analytical superior. Stock solu tions of test lbs had been ready in DMSO or 0. 9% NaCl EGF was dissolved in four mM HCl, and TGFa in four mM HCl containing 1% bovine serum albumin from Sigma Aldrich Cetuximab was dissolved in phosphate buffered saline When answers con taining DMSO had been implemented, the ultimate concentration of DMSO was kept as very low as possible.