Mab 3700

DPCs were lysed and total protein was extracted. Protein concentrations were quantified by the Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific). About 50 μg of extracted protein samples were loaded into each well, separated with 10% SDS-PAGE, then the protein was transferred onto a PVDF membrane. We used 5% non-fat milk in TBST containing Tween-20 to block the membranes at 37°C for 2 h and then incubated them overnight at 4°C supplemented with primary antibodies against BMP2 (1: 200, sc-137087, Santa Cruz Biotechnology, CA, USA), SMAD5 (1: 200, sc-101151, Santa Cruz Biotechnology), p-Smad (1: 1000, mAb #13820, Cell Signaling Technology, Danvers, MA, USA), RUNX2(1: 200, sc-390715, Santa Cruz Biotechnology), DMP-1 (1: 200, sc-73633, Santa Cruz Biotechnology), DSPP (1: 200, sc-73632, Santa Cruz Biotechnology), and ALP (1: 200, sc-373737, Santa Cruz Biotechnology). Membranes were washed with TBST, incubated with IRDye800 conjugated secondary antibody at 37°C for 60 min, and scanned with the Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA). Data were normalized to β-actin (1: 1000, mAb #3700, Cell Signaling Technology) levels and analyzed with Image-Pro Plus software, version 7.0 (Rockville, MD, USA).

Ten micrograms of purified proteins were separated by SDS-PAGE on a 10% Protein gel (Bio-Rad, #4561033) and transferred onto nitrocellulose membranes (0.2 μmol/L, Bio-Rad, 1620252). Membranes were incubated overnight at 4°C with rabbit anti-HA-tag (mAb #3724, Cell Signaling Technology, clone: C29F4, RRID:AB_1549585, 1:1,000 dilution) or Mouse anti-β-actin (mAb #3700, Cell Signaling Technology, clone: 8H10D10, RRID:AB_2242334, 1:2,000 dilution). IRDye 800CW Donkey anti-Rabbit IgG (LI-COR Biosciences, #926–32213, RRID:AB_2715510, 1:15,000) and IRDye 680RD Goat anti-Mouse IgG (LI-COR Biosciences, #926–68070, RRID:AB_2651128, 1:15,000 dilution) were used as secondary antibody. Imaging was performed on the Odyssey CLx Imaging System (LI-COR Biosciences, 9140, RRID:SCR_014579).