(A) Representative images of immunofluorescence analyses of primary osteoblasts transfected with FLAG–pro-OCN or the R46A/R48A/R49A FLAG–pro-OCN mutant (FLAG–pro-OCNAAA) expressed from the p3xFLAG-Myc-CMV-23 vector. Furin is shown in green, FLAG (i.e., pro-OCN) in red, and DNA (DAPI) in blue. Scale bar: 10 μm. (B) Quantification of the red signal area in primary osteoblasts transfected with either FLAG–pro-OCN (n = 9 cells) or FLAG–pro-OCNAAA (n = 7 cells). Results represent the mean ± SEM. *P < 0.05, by unpaired, 2-tailed Student’s t test. (C) Western blot analysis of cell supernatant and cell extract from primary osteoblasts transfected with either FLAG–pro-OCN or FLAG–pro-OCNAAA.