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. 2017 Oct 3;127(11):4104–4117. doi: 10.1172/JCI93437

Figure 5. Pro-OCN processing and γ-carboxylation occur independently of each other in osteoblasts.

Figure 5

(A and B) Western blot analysis of endogenous total OCN (OCN) and γ-carboxylated OCN (Gla OCN) of cell supernatant and cell extract from differentiated mouse osteoblasts treated or not with 50 μM warfarin (A) or 50 μM Dec-RVKR-CMK (RVKR) (B). (C) Western blot analysis of supernatant from osteoblasts transfected with OCN-V5 or the E13D/E17D/E20D OCN-V5 mutant (OCNDDD-V5) and treated or not with 50 μM Dec-RVKR-CMK. (D) Western blot analysis of OCN immunoprecipitated from the serum of control mice (Ggcxfl/fl) and mice lacking γ-carboxylase in osteoblasts (Ggcxosb–/–). Total OCN and γ-carboxylated OCN were assessed by Western blotting. (E) LC-MS/MS analyses of cell supernatant of differentiated mouse osteoblasts treated or not with 50 μM Dec-RVKR-CMK or 50 μM warfarin. Quantification of the OCN propeptide area relative to the total peptide area in 3 independent experiments is shown (see also Table 1). Results represent the mean ± SEM.