Gapdh

Polyclonal antibodies to KIT (western), MAPK, GAPDH and HSP90 were purchased from Dako (Carpinteria, CA), Life Technologies (Carlsbad, CA), Proteintech Group Inc. (Rosemont, IL) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. All phospho-antibodies, polyclonal antibodies to AKT and the monoclonal antibody to S6 were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal mouse antibodies to KIT (co-IP) and CK2 (Santa Cruz Biotechnology, CA), CDC37 (Abcam Biotechnology, Cambridge, MA) and β-actin (Sigma-Aldrich, St. Louis, MO) were used. Anti-mouse normal IgG was purchased from Cell Signaling Biotechnology. CX4945 (CX) and imatinib (IM) were purchased from Selleck (Houston, TX) and LC Laboratories (Woburn, MA, USA), respectively. Lentiviral CK2 shRNA constructs were purchased from The RNAi Consortium (TRC, Cambridge, MA, USA). Crystal violet and propidium iodide solution were purchased from Sigma-Aldrich. Protein A, Protein G beads, Lipofectamine and Plus reagent were purchased from Invitrogen (LIFE Technologies, USA). Puromycin and polybrene were purchased from Sigma (St. Louis, MO, USA).

Proteins were extracted from HTFs using 2 × SDS sample buffer (100 mM Tris HCL pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol and 0.2% bromophenol blue). Equal amounts of protein were loaded onto and run on 4–12% NuPAGE Bis-Tris protein gels (Novex, Life Technologies). The gels were transferred onto nitrocellulose blotting membranes (Amersham, Life Sciences), and blocked in 3% non-fat milk in PBST (PBS 0.1% Tween) for 60 minutes. The membranes were then incubated overnight at 4 °C in primary antibody (MRTF-B, C-19 sc-47282, 1:1000, Santa Cruz; GAPDH, G9545, 1:3000, Sigma). The next day, the membranes were washed three times for 10 minutes each in PBST, and incubated for 1 hour at room temperature in secondary-labelled antibody (MRTF-B: Anti-goat HRP immunoglobulins, 1:2000, Dako; GAPDH: Anti-rabbit HRP immunoglobulins, 1:5000, Dako). The membranes were then washed with PBST three times for 10 minutes each, treated with ECL solution (Amersham, Life sciences) for 5 minutes, and scanned on an Odyssey IR Imager (LI-COR). MRTF-B protein silencing was also measured using densitometric analysis and GAPDH as loading control.