Akt and erk1 2

Cell culture media RPMI 1640 Medium GlutaMAX™, DMEM and M199 were obtained from Life technologies. Recombinant human BMP‐2 and Tumour necrosis factor‐α (TNF‐α), M‐CSF, IL‐1β and IL‐4 were obtained from Peprotech Inc., (Rocky Hill, CT, USA) and human VEGFA from Reliatech. PI3K inhibitor Wortmannin, p38 inhibitor SB239063, and ERK1/2 kinase inhibitor PD98059 and the BMP kinase receptor inhibitor LDN193189 were purchased from Sigma‐Aldrich (Saint Louis, MO, USA). Antibodies recognizing phosphorylated forms of SMAD1 (Ser456/467), AKT (Ser473), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) and total AKT and ERK1/2 were purchased from Cell Signaling Technology Inc., (Danvers, MA, USA). The total p38 and Smad1/5/8 antibodies were obtained from SantaCruz. β‐Actin antibody was obtained from Sigma‐Aldrich.

The following antibodies were used: mouse monoclonal antibodies against CYR61 (A-10), phospho-ERK1/2 (E-4), and β-actin (C4; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); CTGF (Abgent, CA); rabbit monoclonal antibodies against Bcl-xl (54H6), cleaved-PARP (Asp214, #5625), and phospho-AKT (EP2109Y) (Cell Signaling Technology, Inc.); rabbit polyclonal antibodies against CDK6 (C-21), EGFR, phospho-RB (Ser249/Thr252), and YAP (G-6; Santa Cruz Biotechnology, Inc); AKT, and ERK1/2 (Cell Signaling Technology, Inc.).
Western blotting was performed, as previously described^{35 (link)}. In brief, proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and incubated with specific antibodies. The HRP-conjugated secondary antibody was detected by using an enhanced chemiluminescence detection system (Amersham Life Science, NJ, USA) and the ban were imaged by using the Amersham Imager 600 system (GE Healthcare) or X-ray films (Agfa-Gaevert, Mortsel, Belgium).