At this stage of culture, HSCs show phenotypic features of fully activated find more HSC/MFs and a profile of cell surface mark ers identical to that of interface MF described in fibrotic and cirrhotic human livers. HSC/MFs were plated to obtain the desired subconfluence level and then incubated for 24 hours in serum free Iscoves medium in order to obtain cells at the lowest level of spontaneous proliferation before the addition of the dif ferent stimuli. Western blot Cells were lysed with 50 mM 1 piper azineethanesulphonic acid buffer pH 7. 5, 150 mM NaCl, 10% glycerol, 1% Triton X 100, 1. 5 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenylmeth ylsulphonyl fluoride and 100 mM sodium fluoride for 20 minutes at 4 C.

Cells were scraped from dishes and cen trifuged at 15,000 g for 20 minutes at 4 C. Supernatants were loaded for sodium dodecyl sulphate polyacrylamide gel electrophoresis gel. After transferring the proteins, blots were incubated with the desired primary antibodies and then incubated with peroxidase conju gated anti mouse or anti rabbit immunoglobulins in Tris buffered saline Tween containing 1% non fat dry milk and developed with ECL reagents or IMMOBILON Western reagents according to the manufacturers instructions. Akt activity An immune complex kinase assay of Akt activity was per formed as described elsewhere. Briefly, 100 mg of proteins were immunoprecipitated with anti Akt antibod ies followed by adsorption to protein G agarose.

Immu noprecipitates were then collected by a brief centrifugation and washed three times with washing buffer, 40 mM NaCl, 50 mM NaF, 1 mM ethylenediaminetetraacetic acid, 1 mM EGTA, 0. 5% Nonidet P 40, 20 mM b glycerophos phate, 0. 5 mM sodium orthovanadate, 1 mM phenyl methylsulphonyl Brefeldin_A fluoride, 10 mg/ml leupeptin, 10 mg/ ml pepstatin and 10 mg/ml aprotinin. The assay was per formed by resuspending the beads in kinase buffer, 100 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 10 mM b glycerophosphate and 0. 5 mM sodium orthovanadate in the presence of 1 mM protein kinase A inhibitor peptide, 50 mM unlabelled ATP and 6 Ci of ATP, using exogenous histone H2B as the substrate and incubating for 20 minutes at room temperature. Reaction products were run in a 12% SDS PAGE, stained with Coomassie Blue and visualised by autoradiography.

Evaluation of apoptosis Evaluation currently of cell apoptosis was performed by evaluation of PARP and caspase cleavage on Western blot. Statistical analysis All Western blots were representative of at least three to four experiments with similar results. Statistical analysis was performed by students t test. P values 0. 05 or 0. 01 were considered significant. Results In the first set of experiments we investigated the IGF I intracellular signalling downstream of PI 3K activation. As shown in Figure 1, IGF I induced phosphorylation of c Akt on Ser 473 residue after 15 minutes of incubation.