Rabbit anti smad1

Cells were seeded in 24-well plates transfected with empty SLC39A5-FLAG, SLC39A5-M304T-FLAG or SLC39A5-Y46X-FLAG plasmid using Lipofectamine 2000 (Invitrogen). Cells were harvested 48 h after transfection for protein extraction. Cells were lysed and homogenised with SDS (sodium dodecyl sulfate) sample buffer (63 mM Tris-HCl, 10% glycerol, 2% SDS) and Protease Inhibitor Cocktail (Sigma). Twenty micrograms of total protein per lane were separated on 10% SDS-PAGE (polyacrylamide gel electrophoresis) gels and followed by transfer to PVDF membranes (Millipore). Membranes were blocked by 5% non-fat milk for 1 h at room temperature followed by incubation overnight with primary antibody at 4. The antibody used was rabbit-anti Smad1 (Cell Signaling), M2 (Sigma), mouse-anti SLC39A5 (sigma), mouse-anti β-actin (Sigma).

The levels of p-Smad 1/5/9 were analyzed via western blot assay to demonstrate whether CP-BMP2 increases p-Smad 1/5/9 levels in C2C12 murine myoblast cells. After starvation with serum-free medium for 2 h, C2C12 cells (2 x 10⁵ cells per well in 6-well plates) were treated with CP-BMP2 or BMP protein for 2 h at 37 °C. DPBS-washed cells were harvested with RIPA buffer (150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 2 mM EDTA and 50 mM Tris, pH 8.0) containing a protease and phosphatase inhibitor cocktail (Sigma). The quantified cell lysates were separated by 10 % SDS‒PAGE, and the proteins were transferred to nitrocellulose membranes. The antibodies used were as follows: rabbit anti-phosphorylated Smad 1/5/9 (Cell Signaling, #13820), rabbit anti-Smad 1 (Cell Signaling, #6944S), and rabbit anti-β-actin (Cell Signaling, #4967).

Immunoprecipitation of overexpressed proteins from HEK293T cells (24 h posttransfection) and endogenous proteins from MCF7 cells (48 h postseeding) was performed using a modified radio-immunoprecipitation assay buffer (RIPA) freshly supplemented with inhibitors (1 mM PMSF, 2 mM Na₃VO₄, 20 mM Na₄P₂O₇, 50 mM NaF, complete protease inhibitor cocktails [Roche]) as previously described (Benn et al., 2015 (link)). TCLs were taken before incubating the lysates with the indicated primary antibody (1–4 µg/lysate) overnight. rabbit anti-AMOT (TLE) antibody, used for AMOT-BMPR2 interactions, was kindly provided by Lars Holmgren (Karolinska Institutet, Stockholm, Sweden). Furthermore, mouse anti-HA tag (Sigma-Aldrich), rabbit anti-SMAD1 (Cell Signaling Technologies), and rabbit anti-AMOT (BethylLabs) antibodies were used for pull down. Control samples were incubated with either isotype control antibodies or recombinant protein A-Sepharose beads (GE Healthcare). Immunocomplexes were precipitated at 4°C for 2 h with recombinant protein A-Sepharose beads and subsequently washed 3–5× with fresh lysis buffer including inhibitors. Proteins were eluted with 2× Laemmli sample buffer and boiled for 10 min at 95°C prior to Western blotting.