Inducible caBmpr1a Osteoblast Isolation

Generation of inducible caBmpr1a mice was reported previously.^{32 (link),33 (link)}caBmpr1a mice were bred with P0-Cre^{35 (link)} to activate caBmpr1a gene expression in a neural crest-specific manner and primary osteoblasts (both caBMPR1A^Q233D and wild-type) were harvested from frontal bones of newborn mouse calvaria.^{33 (link)} Osteoblasts were cultured in alpha-minimum essential medium (αMEM) supplemented with 10% fetal bovine serum (ATCC, Manassas, VA, 30–2020) and 1% penicillin/streptomycin (ATCC, 30–2300). When required, cells were passaged with 0.25% trypsin EDTA (Life Technologies, Carlsbad, CA, 25200–056). All cells used for experiments were at passage 4 or lower. To evaluate levels of BMP-Smad signaling, isolated osteoblasts were cultured with or without rhBMP2 at 100 ng/mL for 30 min. Levels of phospho-Smad1/5/8 were measured by Western blot using a rabbit antiphospho-SMAD1/5/8 (pSMAD1/5/8) (1:1000, 9511, Cell Signaling) (Figure S1).