Nitrocellulose

Total cell protein was recovered using M-PER containing Halt Protease and Halt Phosphatase Inhibitor Cocktails and quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Wilmington, DE, http://www.fishersci.com/). Proteins were electro-phoresed through 10% SDS-polyacrylamide gels and transferred to nitrocellulose (Invitrogen). Membranes were blocked in 5% milk and incubated with primary antibodies against: phosphorylated Smad1/5/8 (1:750) and β-actin (1:3,000) (Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com/), at 4°C overnight. Bound antibodies were detected with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:6,000) (Cell Signaling Technology) at room temperature, 1 hour. Detected proteins were imaged with Immobilon Chemiluminescent HRP Substrate (Millipore, Billerica, MA, http://www.millipore.com) and quantified using ImageJ Software.

C2C12 cells were plated into 6-well plates (2.5×10⁵ cells/well) and cultured overnight at 37°C. Cells were transfected with V5- or HA-tagged ACVR1 constructs as described above. For stimulation with BMP4, cells were serum-starved (0.5% FBS in DMEM) for 2 hours followed by treatment with 30ng/ml BMP4 (R&D Systems) for 1 hour. Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with Halt Protease and Halt Phosphatase Inhibitor Cocktail (Pierce), and cleared by centrifugation. Protein concentrations of lysates were quantified by BCA Protein Kit (Thermo Scientific). Proteins were electrophoresed through 10% Tris-Glycine gels (Invitrogen), and transferred to nitrocellulose (Invitrogen). Membranes were blocked in 5% milk in 1xTBS (BioRad; #1706435) and incubated overnight at 4°C with primary antibodies against phospho-Smad1/5/8 (Cell Signaling Technology, #9511), V5 (Invitrogen R960-25), HA (Sigma Aldrich, #H6908) or β-actin (Cell Signaling Technology, #4967) followed by detection using an anti-rabbit (Cell Signaling Technology, #7074) or anti-mouse (Santa Cruz, #H2208) HRP-conjugated secondary antibody. Membranes were incubated with HRP substrate (LI-COR) and chemiluminescence was detected and quantified with a C-DiGit Blot Scanner (LI-COR).

Naive WT CD4⁺ cells were stimulated with plate-bound anti-CD3ε and anti-CD28 (10 μg/ml each) in the presence of anti-mouse IL-2 for 72 hours and then recultured with human IL-2 for 24 hours (100 U/ml), rested for 12 hours, and stimulated with either anti-CD3ε and anti-CD28 or IL-2 for the indicated tie points. Cells (10⁷) were then lysed in buffer containing 50 mM Hepes (pH 7.4), 150 mM NaCl, 20 mM NaF, 4 mM EDTA, 50 μM sodium orthovanadate, small peptide inhibitors, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5% (v/v) Triton X-100 (Roche, CH). Protein was precipitated with cold acetone, resuspended in 2× Laemmli reducing sample buffer, separated on a 4 to 12% SDS–polyacrylamide gel electrophoresis gel, and Western blotted onto nitrocellulose (Invitrogen or Bio-Rad, USA). Proteins were identified using anti–p-STAT5 (clone A-9, Santa-Cruz Biotechnologies, USA), p-Thr³⁰⁸ PKB/Akt (catalog 9275, Cell Signaling Technologies, USA), p-Thr²⁴–Foxo1 (catalog 2599, Cell Signaling Technologies), or β-actin (catalog 3700T, Cell Signaling Technologies).