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. 2011 May;90(5):619–624. doi: 10.1177/0022034510397839

Figure 1.

Figure 1.

Detection of DMP1 in NCP extracts of dentin from six-week-old WT, Dmp1-KO, Dmp1-KO/D213A-Tg, and Dmp1-KO/normal-Tg mice. (A) We performed Stains-All staining to visualize the DMP1-related components and other NCPs. NCPs were extracted from the dentin of six-week-old WT, Dmp1-KO, Dmp1-KO/D213A-Tg, and Dmp1-KO/normal-Tg mice. The numbers on top of each figure represent the anion-exchange chromatographic fractions. A 60-µL quantity of sample from every third fraction between 44 and 53 was loaded onto SDS-PAGE. DMP1-N and DMP1-C fragments (black arrowheads) were observed in the dentin extracts from WT and Dmp1-KO/normal-Tg mice. Full-length DMP1 (between the 95 kDa and 118 kDa molecular-weight markers; indicated by black arrows) was clearly detected in the dentin extract from the Dmp1-KO/D213A-Tg mice; our previous work (Huang et al., 2008) showed that full-length DMP1 migrates at this position. The identification of this protein band as DMP1 was further confirmed by Western immunoblotting (see below). A weak protein band representing full-length DMP1 was also seen in the dentin extract from the Dmp1-KO/normal-Tg mice. The broad protein band migrating just below the 95-kDa molecular marker in fractions 50 and 53 represents dentin phosphoprotein (DPP, red arrowhead), which co-eluted with DMP1 in these fractions. DPP could be considered an internal control, showing that the total amount of proteins loaded onto each SDS-PAGE well was similar among the different groups. (B) Western immunoblotting was performed with the anti-DMP1-C-857 antibody. The volume and identities of the samples were the same as in Stains-All staining (Fig. 1A). Ctrl: 2 µg of DMP1 (including full-length and fragments) isolated from rat long bone was used as a positive control (ctrl). The protein bands migrating between the 95-kDa and 118-kDa molecular markers (black arrow) represent the full-length form of DMP1. The arrowheads indicate DMP1-C (~57 kDa). Only DMP1-C was observed in the WT mice. No protein bands were detected in the Dmp1-KO mice. Only the full-length form of DMP1 was detected in the Dmp1-KO/D213A-Tg mice when this volume (60 µL) of sample was loaded. Both the full-length and fragment forms of DMP1 were detected in the Dmp1-KO/normal-Tg mice. The above findings indicate that normal DMP1 was cleaved into fragments, whereas D213A-DMP1 was not.