Phosphorylated smad1 5 8

MC3T3-E1 cells were transfected with siRNA targeting APPL1 or scrambled control siRNA using Lipofectamine in Opti-MEM (Invitrogen) according to the manufacturer’s recommendations. Transfected cells were used 48 hours later. Polyacrylamide gel electrophoresis and Western blot analyses were performed essentially as previously described [56 ]. Antibodies for β-actin (1:10000), phosphorylated Smad1/5/8 (1:1000), and APPL1 (1:2000) were purchased from Cell Signaling (Danvers, MA). Antibodies for total Smad1/5/8 (1:500) and AdipoR1 (1:250) were purchased from Santa Cruz Biotechnology. The antibody to detect CK2β (1:5000) was purchased from Bethyl Laboratories (Montgomery, TX, USA). The secondary antibodies were horseradish peroxidase-linked goat-anti-rabbit IgG (Santa Cruz Biotechnology). Blots were visualized using SuperSignal West Dura Substrate (Thermo Scientific, Billerica, MA).

ACVR1 mutations R206H and G328E were cloned into pcDNA3.1 by site-directed mutagenesis as previously described^{16 (link)} and transfected into primary cells QCTBR059 and SU-DIPG-VI using lipofectamine (Invitrogen), with protein collected after 24 hours using standard procedures. Western blots were carried out for anti-FLAG HRP (#A8592, Sigma; 1:1000 dilution) and phosphorylated Smad1/5/8 (#9511, Cell Signalling; 1:1000) under standard conditions. Relative levels of phosphorylated Smad1/5/8 were measured by Image J software (National Institute of Mental Health, Bethesda, MD, USA).