Cell Lysis and Immunoblotting Protocol

Cell lysis and immunoblotting were essentially performed as described previously [13 (link),18 (link),19 (link)] with minor modifications. Proteins were fractionated by polyacrylamide gel electrophoresis on TGX Stain-Free FastCast gels (BioRad, Munich, Germany). Following blotting and antibody treatment, chemoluminescent detection of proteins was done on a ChemiDoc XRS imaging system (BioRad, Munich, Germany) with an Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The antibodies used were: ALK2: Biorbyt Ltd., Cambridge, UK; phospho-SMAD1/5 and SMAD1: Cell Signaling Technology (CST), Frankfurt/Main, Germany; RUNX3/AML2: D9K6L, CST; RAC1: #610650, BD Biosciences, Heidelberg, Germany; RAC1b: #09-271, Merck Millipore, Darmstadt, Germany; GAPDH: 14C10, CST; HSP90: #13119, Santa Cruz Biotechnology, Heidelberg, Germany. The signals for the proteins of interest were normalized to the total amount of protein in the same lane, and significant differences (p < 0.05) were calculated with the unpaired two-tailed Student’s t test.