Phospho smad

Antibodies monoclonal against Ac-Tub and α-SMA were from Sigma-Aldrich (Saint Quentin Fallavier, France), rabbit against Kif-3A was from Abcam (Cambridge, UK) rabbit anti-AKT, phospho-Akt (thr 308), Smad, phospho-Smad, ERK, phospho-ERK were from Cell Signaling (Ozyme, St Quentin en Yvelines, France), monoclonal anti ARL13B (sc-515784) was from Santa Cruz (Ozyme, St Quentin en Yvelines, France), rabbit anti pericentrin was from Bethyl (Euromedex, Souffleweyrsheim, France). Antibody against mouse coupled to Alexa Fluor 488 and antibody against rabbit coupled to Alexa fluor 647 were from Life Technologies (Saint Aubin, France). Antibodies against CD56 APC and CD140a PE were purchased from BD-Biosciences (Le Pont de Claix, France). HPI-4 was from Sigma-Aldrich. Shh-conditioned medium was obtained from an HEK 293 cell line stably transfected with Shh-N expression vector (ATCC #CRL-2782).

DMEM for cell culture was purchased from Corning (Corning, NY, USA). Fetal bovine serum, protease inhibitor cocktail, and αSMA antibody were from Sigma-Aldrich (St. Louis, MO, USA). IGF1R inhibitor I-OMe-Tyrphostin AG 538 was from Calbiochem (San Diego, CA, USA). Recombinant human IGF-II and anti-IGF-II antibody were purchased from R&D Systems (Minneapolis, MN, USA). Collagen, Fibronectin, and GAPDH antibodies were from Santa Cruz (Santa Cruz, CA, USA). IGF1R, IGF2R, IR-β, phospho-SMAD and total SMAD antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine^® 2000 was purchased from Invitrogen (Carlsbad, CA, USA). IGF1R, IR, and isotype control antibodies used in neutralization experiments were obtained from GroPep Bioreagents (Thebarton SA 5031, Australia). IGF1R, IGF1R, IR, and scrambled siRNA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HRP-conjugated anti-mouse IgG was from Promega (Madison, WI, USA) and anti-rabbit-HRP IgG was from GE Healthcare Life Sciences (Chicago, IL, USA).

Cultured cells were lysed in extraction buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, and protease inhibitor cocktail] and quantified using a BCA Protein Assay kit (Pierce, Rockford). Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Membranes were incubated with appropriate primary antibodies against Flag, actin (Sigma-Aldrich), Runx2 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Smad, Smad, phospho-p38, p38, phospho-JNK, JNK, phospho-Erk, Erk (Cell Signaling Technology, Beverly, MA), and lamin B1 (Abchem, Cambridge, UK). Following washing and incubation with appropriate horseradish peroxidase-linked secondary antibodies, signals were detected with an LAS3000 luminescent image analyzer (GE Healthcare, Piscataway, NJ).