Figure 6
Ucma/GRP is involved in suppressing osteo/chondrogenic differentiation by interacting with BMP-2. (A) Quantification of calcification deposited by mVSMCs incubated with 100 ng/ml noggin in the presence of osteogenic medium (2.6 mM phosphate) for 12 days, n = 6. Statistical significance was assessed using ANOVA. (B) Conditioned media from hVSMCs treated with 500 ng/ml of BMP-2 for 24 h was used to capture BMP-2-containing protein complex by Co-immunoprecipitation (Co-IP) with a BMP2/4 antibody, and the presence of BMP2 and GRP in the eluted complex was detected by western blot. Two bands (25 kDa and 15 kDa) correspond to the dimeric and monomeric BMP-2/4. Representative image of 3 independent experiments. (C) Quantification of calcification deposited by mVSMCs from Ucma/GRP KO and WT mice, n = 8. Cells were incubated with 2 µM dorsomorphin in osteogenic medium (OM) for 12 days. Statistical significance was assessed using ANOVA. (D) Western blotting analysis of phosphorylated SMAD 1/5/8 and ALP and in Ucma/GRP−/− mVSMCs treated with 2 µM dorsomorphin in osteogenic medium (OM) for 12 days. Figure shows full western blots obtained by cutting of a full-sized membrane prior to antibody incubations. Numbers in the middle show molecular weights of the protein standard. Numbers on the right show the expected molecular weights of the proteins. (E,F) Quantification of Western blots from panel C, n = 3. Statistical significance was assessed using t-test. All graphs show mean + SD. *p between 0.05–0.01, **p between 0.001–0.01, ***p < 0.001.
