Figure 3.

Western immunoblotting for the SIBLING family members in the extracts from the predentin/dentin complex and the long bone of WT and Hyp mice. A representative fraction is shown, to illustrate the Western immunoblotting results for the SIBLING members. As appropriate antibodies against DPP are not available, we could not carry out Western immunoblotting for DPP. (a–f) Results for the SIBLING members extracted from the predentin/dentin complex. (g–i) Results for the SIBLING members extracted from the long bone. (a, c) Western immunoblotting using the anti-DMP1-N-terminal-9B6.3 monoclonal Ab. Positive control (Ctrl): 1 μg of the N-terminal fragment of DMP1. The core protein of the DMP1-N terminal fragment (37 kDa) (a) and DMP1-PG (c) were detected by the anti-DMP1-N-terminal-9B6.3 Ab. Although there was no difference for the core protein of the DMP1 N-terminal fragment (37 kDa) between the WT and Hyp mice, DMP1-PG was more abundant in the predentin/dentin of the Hyp mice. (b) Western immunoblotting using the anti-DMP1-C-terminal-857 polyclonal Ab. Ctrl: 1 μg of COOH-terminal fragment of DMP1. Note significant difference in the quantity was observed for the DMP1 C-terminal fragment between the WT and Hyp mice. (d) Western immunoblotting using the anti-DSP polyclonal Ab. Ctrl: 0.5 μg of DSP isolated from rat dentin. The expression level of DSP in the dentin of the Hyp mice was similar to WT mice. (e) Western immunoblotting using the anti-BSP-10D9.3 monoclonal Ab. Ctrl: 0.5 μg of BSP isolated from rat long bone. The quantity of BSP in the predentin/dentin of Hyp mice was similar to WT mice. (f) Western immunoblotting using the anti-OPN monoclonal Ab. Ctrl: 1 μg of OPN isolated from rat long bone. The quantity of OPN in the predentin/dentin of Hyp mice was similar to WT mice. (g) Western immunoblotting using the anti-DMP1-N-terminal-9B6.3 monoclonal Ab. Ctrl: 1 μg of DMP1 isolated from the rat long bone. The extracellular matrix (ECM) of the long bone of the Hyp mice had more DMP1-PG than in the WT mice. (h) Western immunoblotting using the anti-BSP-10D9.3 monoclonal Ab. Positive control (Ctrl): 0.5 μg of BSP isolated from the rat long bone. The ECM of the long bone of the Hyp mice had more BSP than the WT mice. (i) Western immunoblotting using the anti-OPN monoclonal Ab. Ctrl: 1 μg of OPN isolated from the rat long bone. The ECM of the long bone of the Hyp mice had more OPN than the WT mice. (j) Quantitative analyses of Western immunoblotting results for SIBLING proteins extracted from the dentin and bone of Hyp and WT mice. For normalization, the quantity of any of the SIBLING proteins from the WT was set as 1, while the quantity of each protein from the Hyp mice was expressed as relative folds to that from the WT mice. The amount of DMP1-PG in the Hyp mouse dentin was more than 12-fold greater than the WT mice. Also note that there was more than eight times of BSP in the Hyp mouse bone than in the WT. The data represent calculations from three separate Western immunoblots that agreed closely