The Src inhibitor CGP77675 was a gift from Novartis Pharma AG (Ba

The Src inhibitor CGP77675 was a gift from Novartis Pharma AG (Basel, Switzerland). All other chemicals were of analytical quality. Antibodies against phosphorylated AktSer473, total Akt, dually phosphorylated sellekchem ERKThr202/Tyr204, GAPDH and phospho-ShcTyr239/240 were obtained from Cell Signaling Technology (Boston, MA). Antibody against phospho-EGF receptorTyr1173 was obtained from Invitrogen. Anti-ERK antibody was from Upstate/Millipore (Billerica, MA). Secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA) and Licor Biosciences (Lincoln, NE). Cells and culturing The rat hepatocarcinoma cell line MH1C1, derived from a Morris hepatoma [39], was obtained from ATCC (Manassas, VA). The cells were seeded onto Costar plastic flasks and cultured in Dulbecco��s Modified Eagle��s medium.

The Medium was supplemented with horse serum (10%), glutamine (2mM), and 100 U/ml Pen-Strep. The cultures were kept in a humidified 5% CO2 incubator at 37��C. Cells were seeded onto culture wells at a density of 40 000�C50 000 cells per cm2. After 24hours, the medium was changed and the cells were cultured under serum-free conditions 24h prior to stimulation. Hepatocytes were isolated from male Wistar rats as previously described [40]. The hepatocytes were seeded onto Costar plastic culture wells at a density of 15 000�C20 000 per cm2. The culture medium was a serum-free 1:1 combination of William��s Medium E and Dulbecco��s Modified Eagle��s Medium. The medium was supplemented with 100 U/ml Pen-Strep, collagen (3��g/ml), insulin (100 nM) and dexamethasone (25 nM).

Immunoblotting Aliquots containing ~30000 MH1C1 cells or hepatocytes (total cell lysate prepared in Laemmli or RIPA buffer) were electrophoresed on 6�C12% (w/v) polyacrylamide gels (acrylamide: N��N��-bis-methylene acrylamide 30:1). This was followed by protein electrotransfer to nitrocellulose membranes and immunoblotting with antibodies against proteins as described in the figures. Usually the same membrane was stripped and reincubated with different antibodies, and then one single loading control was used as the final incubation. Immunoreactive bands were visualized with enhanced chemiluminescence using LumiGLO (KPL Protein research Products, Gaithersburg, MD) or by infrared imaging using Odyssey Infrared Imaging System, supplied by Licor Biosciences (Lincoln, NE).

RNA isolation and cDNA synthesis RNA from MH1C1 cells was isolated with Qiagen RNeasy kit according to the manufacturer��s instructions, and was treated with DNAse. The integrity of RNA was evaluated by ethidium bromide agarose Batimastat gel electrophoresis, and the quantity and purity was measured spectrophotometrically (OD 260/280). cDNA was synthesized from 1.0��g RNA with Superscript? III reverse transcriptase (Invitrogen) according to manufacturer��s protocol.

Leave a Reply

Your email address will not be published. Required fields are marked *


You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>