antibodies against ambn  (R&D Systems)

Journal: Scientific Reports

Article Title: Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

doi: 10.1038/s41598-023-28388-4

Figure Lengend Snippet: Schemes of unrooted, majority consensus trees of four major enamel matrix proteins. General phylogenetic consensus ( www.timetree.org 2021/07/27) as a scheme in the center ( a ) and schematic phylograms for Amelogenin (AMELX— b ), Ameloblastin (AMBN— c ), Amelotin (AMTN— d ), and Enamelin (ENAM— e ) are displayed highlighting clustering of protein sequences to higher taxonomical groups. Enamel- and toothless species in red, the most basal species (i.e. Latimeria or Polypterus ) in blue. Clusters in bracelets or with a Roman number (I, II, III) are not homogenous in the analysis. There is great stability not only of major taxonomic groups and general tree topology, but also the absence of enamel does not influence the position of the species. Colors correspond to taxonomic clades: Amphibia (dark grey), Lepidosauria (green), Archosauria (dark green); Marsupialia (sea blue), Afrotheria (pink), Primates (red), Lagomorpha (dark blue), Rodentia (blue), Eulipotyphla (light blue), Chiroptera (yellow), Carnivora (light green), Perissodactyla (violet), “Artiodactyla” (brown), Odontoceti (light brown). Full phylograms are in Supplementary Fig. 1–4.

Article Snippet: Then, antigen retrieval was performed for 5 min in the pressure cooker with HIER Citrate Buffer pH 6.0 (Zytomed, Germany), the sections were blocked by 10% normal goat serum (ThermoFisher Scientific, USA) for 1 h at room temperature, and incubated overnight with primary antibodies against AMBN (diluted 1:100, AF3026, RandD Systems, USA) or AMELX (diluted 1:200, produced in Wald et al., 2017).

Techniques:

Journal: Scientific Reports

Article Title: Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

doi: 10.1038/s41598-023-28388-4

Figure Lengend Snippet: Identification of functional motives of EMPs. Detected functional motifs of AMELX ( a ), AMBN ( b ), AMTN ( c ), and ENAM ( d ). N-terminus to left, C-terminus to right. The position and motif structure corresponds to Mus musculus sequences for these proteins (AAB93765.1, BAA06546.1, NP_082069.1, and AAB94312.1). Color of the motif corresponds to the motif type (CLV = cleavage, DEG = degradation, DOC = docking, LIG = ligand, MOD = modifier, TRG = transport), while saturation to the minimal age of the motif, i.e. for how large taxonomical unit (Euarchontoglyres < Placentalia < Mammalia < Amniota < Tetrapoda < Sarcopterygii/Euteleostomi) is it common. The self-assembly motifs are highlighted in the AMELX (Y–L–Y; and Y–Y–Y) and AMBN (Y–L–F) structure. Detailed information about motifs in Supplementary table 1.

Article Snippet: Then, antigen retrieval was performed for 5 min in the pressure cooker with HIER Citrate Buffer pH 6.0 (Zytomed, Germany), the sections were blocked by 10% normal goat serum (ThermoFisher Scientific, USA) for 1 h at room temperature, and incubated overnight with primary antibodies against AMBN (diluted 1:100, AF3026, RandD Systems, USA) or AMELX (diluted 1:200, produced in Wald et al., 2017).

Techniques: Functional Assay

Journal: Scientific Reports

Article Title: Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

doi: 10.1038/s41598-023-28388-4

Figure Lengend Snippet: Functional pleiotropy of EMPs revealed by unbiased phenotyping. Circular graphs visualizing significant difference of mouse mutants from WT baseline divided to 8 groups phenotypes: sensory (e.g. hearing or eye retina morphology—in blue-green), morphology (skeletal morphology and bone mineralization—in orange), cardiovascular (e.g. blood pressure, cardiography—in violet), metabolism (e.g. body mass, lean/fat ratio, glucose tolerance—in magenta), immunology (T-cells and B-cells populations characterization—in green), behavioral (e.g. grip strength, dark/light box test, hot/cold plate test—in yellow), reproduction (i.e. number of litters—in brown), and respiratory (e.g. capacity and elasticity of lungs—in gray). Each radius is one variable. Thick black circle marks the level of significance = 0.05—points outside the circle are significant results. The massive impact of all mutations on various levels of phenotype was evident. Amelx KO, Ambn KO, and Ambn G/G , and were phenotyped in the Czech Centre for Phenogenomics (Vestec, Czech Republic), Amtn KO, and Enam KO in UC Davis (California, USA). Overview of statistical results is in Supplementary table 2.

Article Snippet: Then, antigen retrieval was performed for 5 min in the pressure cooker with HIER Citrate Buffer pH 6.0 (Zytomed, Germany), the sections were blocked by 10% normal goat serum (ThermoFisher Scientific, USA) for 1 h at room temperature, and incubated overnight with primary antibodies against AMBN (diluted 1:100, AF3026, RandD Systems, USA) or AMELX (diluted 1:200, produced in Wald et al., 2017).

Techniques: Functional Assay

Journal: Scientific Reports

Article Title: Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

doi: 10.1038/s41598-023-28388-4

Figure Lengend Snippet: Bone ultrastructure is affected in Ambn and AmelX KO. ( a ) Trabecular structure. Pseudocolors correspond to trabecular thickness (spectrum on the right). Bar = 0.5 mm. Specimens closest to group’s average selected. ( b ) Whisker plots of femoral cortical bone microstructure: total volume of cortical bone (including pores), total volume of pore space, both in mm3, and mineralization of cortical bone in Attenuation units (AU). ( c ) Whisker plot of femoral trabecular bone: mean number of trabeculae per section, the mean thickness of trabeculae in mm, and mineralization of trabecular bone in AU. Females on the left and males on the right of each graph. Thick midline = mean, whiskers = standard deviation; * < 0.05; ** < 0.01, *** < 0.001. Values for each observation are shown as points—WTs in red, Ambn KO in green, Ambn G/G in blue, and Amelx KO in violet.

Article Snippet: Then, antigen retrieval was performed for 5 min in the pressure cooker with HIER Citrate Buffer pH 6.0 (Zytomed, Germany), the sections were blocked by 10% normal goat serum (ThermoFisher Scientific, USA) for 1 h at room temperature, and incubated overnight with primary antibodies against AMBN (diluted 1:100, AF3026, RandD Systems, USA) or AMELX (diluted 1:200, produced in Wald et al., 2017).

Techniques: Whisker Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions

doi: 10.1038/s41598-023-28388-4

Figure Lengend Snippet: Long bone morphology and strength in Ambn and AmelX mutant mice. ( a ) Structure of femur—bone morphology (35 µm voxel resolution) on the left and porosity of the cortical bone (1.5 µm voxel resolution) on the right. Pseudo colors correspond to differences in mineralization in the whole femur (blue–yellow spectrum), or the pore size in the section (violet–red spectrum). Blue vertical bar (whole femur) = 2 mm, white horizontal bar (section with pores) = 0.5 mm. ( b ) Whisker plots of femur morphometry and bone fracture analysis characteristics: femur length, femur width (both in mm), and bone strength (in Newtons, N). Females on the left and males on the right of each graph. Thick midline = mean, whiskers = standard deviation; *< 0.05, ***< 0.001. Values for each observation are shown as points—WTs in red, Ambn KO in green, Ambn G/G in blue, and Amelx KO in violet.

Article Snippet: Then, antigen retrieval was performed for 5 min in the pressure cooker with HIER Citrate Buffer pH 6.0 (Zytomed, Germany), the sections were blocked by 10% normal goat serum (ThermoFisher Scientific, USA) for 1 h at room temperature, and incubated overnight with primary antibodies against AMBN (diluted 1:100, AF3026, RandD Systems, USA) or AMELX (diluted 1:200, produced in Wald et al., 2017).

Techniques: Mutagenesis, Pore Size, Whisker Assay, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: FAM20C Plays an Essential Role in the Formation of Murine Teeth

doi: 10.1074/jbc.m112.386862

Figure Lengend Snippet: FIGURE 7. Ameloblastin and amelotin are down regulated in Fam20C-deficient ameloblasts. A and B, ISH of AMBN on sagittal sections of the lower incisors from newborn mice. The incisor of WT mice (left) showed strong AMBN mRNA staining (red) in ameloblasts (arrow), whereas the incisor of KO mice (right) had remarkably less AMBN in the ameloblasts (arrow). C and D, IHC of ameloblastin protein revealed remarkably less AMBN (brown, arrow) in the ameloblasts of the incisor in the KO mice (right) compared with the WT mouse incisor (left). E and F, ISH of AMTN revealed remarkable down-regulation of AMTN in the mature ameloblasts (arrow) of the incisor in the KO mice (right) compared with the strong staining in the mature ameloblasts (red, arrow) of the WT mouse incisor (left). G–P, ISH of AMEL, ENAM, MMP20, KLK4, and ODAM showed no significant differences in the expression levels between KO (right) and WT mouse teeth (left). Scale bars, 200 m.

Article Snippet: A polyclonal antibody against ameloblastin (Santa Cruz Biotechnology, Inc.) was employed following the manufacturer’s instructions.

Techniques: Staining, Expressing