Results: 3

Figure 3.

Figure 3. From: Msx1 Mutations.

Luciferase reporter assays of synergistic promoter up-regulation. (A) Bmp4 promoter-reporter constructs (p2.4 Bmp4-luciferase) or (B) Msx1 promoter-reporter constructs (p3.5 Msx1-luciferase) were co-transfected with the different Msx1 expression vectors and the wild-type Pax9 expression vector at a 1:2 ratio. (Error bars represent standard error; *p < 0.05, **p < 0.01.)

Y. Wang, et al. J Dent Res. 2011 March;90(3):311-316.
Figure 1.

Figure 1. From: Msx1 Mutations.

Sequence alignments and 3D structure prediction. (A) Protein sequence alignment of human MSX1/2 and mouse Msx1/2/3. The positions of mutated amino acids are indicated with grey shading. The secondary structure of the Msx1 homeodomain is shown above the sequence. Two of the mutations are located within helix 2 and the other two in helix 3. (B) Protein-DNA interaction models of the Msx1 homeodomain with and without the missense mutations. The homeodomain protein chain is red, and the DNA double helix is shown in blue. The wild-type and mutant amino acids stand out as yellow stick models.

Y. Wang, et al. J Dent Res. 2011 March;90(3):311-316.
Figure 2.

Figure 2. From: Msx1 Mutations.

Characterizations of Msx1 protein. (A) Msx1 protein half-life: Cos7 cells were transfected with Flag-Msx1 expression vectors and treated with 90 mM cycloheximide (CHX) for 1 to 6 hrs. Western blot of cell lysates with anti-FLAG was used to assess the protein degradation speed; actin served as a control. (B) DNA-binding abilities: Electrophoretic mobility shift assay (EMSA) was used to test DNA-binding activity of nuclear extracts from Flag-Msx1-transfected COS7 cells with a double-stranded oligonucleotide probe containing a consensus binding-site for Msx1. Specificity of the complexes was confirmed by super-shift induction with anti-FLAG antibody. To verify that equal amounts of Msx1 protein were used for the EMSA, we performed a Western blot of nuclear extracts (lower panel). (Note that R196P migrates more slowly than the other proteins.) (C) Nuclear localization: YFP-Msx1 fusion proteins were expressed in COS7 cells. None of the missense mutations affected nuclear localization, in contrast to the YFP-Msx1 construct without homeodomain, which is found in the cytoplasm (YFP-delHD). The homeodomain by itself suffices for translocation to the nucleus (YFP-HD). YFP-R196P showed densely clumped distribution within the nuclei. These protein aggregates could be the consequence of protein interactions or of a secondary modification which also causes the R196P mutant to migrate more slowly than the other mutants and wild type Msx1. (D) Protein interaction: Co-immunoprecipitation with COS7 cells co-transfected with Myc-tagged Pax9 proteins and FLAG-tagged Msx1. Upper panel: Western blot analyses of immunoprecipitates of whole-cell lysates pulled down with anti-FLAG antibody and probed with an anti-Pax9 antibody. Middle panel: Whole-cell lysates probed with anti-Pax9 antibody or (lower panel) anti-Flag antibody. (Note that the Msx1-R196P migrates more slowly in both immunoprecipitations and input cell lysate.)

Y. Wang, et al. J Dent Res. 2011 March;90(3):311-316.

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