Figure 3

Predominant expression of obif protein in osteoblast-lineage cells. (A-E) Immunostaining of developing limbs using anti-obif antibody. Sections of limbs were stained with anti-obif antibody (red) and nuclei were stained with DAPI (blue). E15.5 forelimb sections (A, B). Scale bars = 200 μm. E17.5 humerus sections (C). Obif was expressed in spindle-shaped osteoblasts lining trabecular bone surfaces and in cuboidal-shaped osteoblasts. Scale bars = 50 μm. Higher magnification of E17.5 humerus sections shows that obif is localized to the plasma membrane (D, E). White arrows indicate typical cells that show the transmembrane pattern. (F, G) E 18.5 femur section was first stained with anti-obif antibody (green) and then stained for ALP. (H, I) E 18.5 hindlimb section was double-stained with anti-obif antibody (red) and anti-collagen I antibody (green). (J-O) Primary calvarial cells cultured in 12-well plates were immunostained with anti-obif antibody (J-L) and DAPI (M-O). Sparsely cultured cells (J, M). Densely cultured cells were incubated in presence of ascorbic acid for 23 days and used for analysis (K-O). L & O are negative controls. (P) Detection of endogenous obif protein. Whole cell lysates of calvarial cells-obif (stably infected), calvarial cells-day 0 (not stimulated), calvarial cells-day 23 (stimulated for 23 days), MC3T3-E1 cells-day 0 (not stimulated), MC3T3-E1 cells-day 8 (stimulated for 8 days), and MC3T3-E1 cells-day 14 (stimulated for 14 days) were electrophoresed by SDS-PAGE. Immunoblots were probed with the anti-obif antibody. Calvarial cells-obif is a positive control.