Smad2 3 antibody

LβT2 cells were incubated overnight in serum-free media and then treated with or without 10 ng/mL activin for 2 hours. The cells were harvested and nuclear extracts were prepared, as previously described [49] (link). Protein concentration was determined by Bradford assay. 400 µg of pre-cleared nuclear extracts were incubated with 4 µg of mouse IgG (Santa Cruz sc-2025), SMAD4 antibody (sc-7966) or SMAD2/3 antibody (BD Biosciences 610842) at 4°C for 1 hour. Twenty-five µL of Protein A Magnetic Beads (New England Biolabs, Ipswich, MA) were added and the extracts were rocked overnight at 4°C. Bead/protein complexes were washed 1× with PBS then eluted in 2× SDS sample buffer at 70°C for 5 minutes. 20 µg of protein was electrophoresed on a 10% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane and blocked overnight in 5% non-fat dry milk in 1× Tris-buffered saline with 0.1% Tween-20. The blots were then incubated overnight at 4°C with rabbit anti-SMAD4 (Millipore 04-1033; 1∶1000 dilution), SMAD2/3 (sc-8332; 1∶1000) or FOXO1 (sc-11350, 1∶1000 dilution) primary antibodies. Blots were incubated with a goat anti-rabbit horseradish peroxidase-linked secondary antibody (Santa Cruz; 1∶5000) and bands were visualized using the SuperSignal West Dura Substrate (Thermo Scientific).

Recombinant human M-CSF was purchased from R&D Systems (Minneapolis, MN, USA), and TGF-β and SB431542 were from Sigma-Aldrich (St Louis, MO, USA). GST-RANKL was purchased from Oriental Yeast Co., Ltd (Shiga, Japan). Alpha-minimum essential medium (α-MEM) and fetal bovine serum (FBS) were purchased from Life Technologies (Carlsbad, CA, USA). Smad2/3 antibody was purchased from BD Biosciences (Monoclonal antibody, Mouse, 610843, San Jose, CA, USA), anti-trimethyl-histone H3 lysine 4 was from Activemotif (Polyclonal antibody, Rabbit, 39159, Carlsbad, CA, USA), anti-trimethyl-histone H3 lysine 27 was from Millipore (Polyclonal antibody, Rabbit, 07–449, Billerica, MA, USA), anti-β-actin was from Sigma-Aldrich (Polyclonal antibody, Rabbit, A2066, St Louis, MO, USA).