pax9  (OriGene)

Journal: Cell death and differentiation

Article Title: Genome-wide screening for deubiquitinase subfamily identifies ubiquitin-specific protease 49 as a novel regulator of odontogenesis.

doi: 10.1038/s41418-022-00956-7

Figure Lengend Snippet: Fig. 2 USP49 upregulates endogenous PAX9 and MSX1 proteins. A Validation of the efficiency of sgRNAs were carried out in hDPSCs by transiently transfecting sgRNA1 to sgRNA4 targeting USP49, and immunoblotting with the USP49 antibody. The presented immunoblots are representative of three independent experiments (n = 3). B Validation of the efficiency of shRNAs were carried out in hDPSCs by transiently transducing shRNA1 and shRNA2 targeting USP49, and immunoblotting with the USP49 antibody. The presented immunoblots are representative of three independent experiments (n = 3). C hDPSCs were transfected with two best sgRNAs (sgRNA1 and sgRNA3) and one shRNA (shRNA1) targeting USP49 to check the endogenous protein levels of USP49, PAX9, and MSX1 using their respective endogenous antibodies. The presented immunoblots are representative of three independent experiments (n = 3). D hDPSCs were transfected with increasing concentrations of Flag-USP49 to check the endogenous USP49, PAX9, and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). E hDPSCs were transfected with increasing concentrations of Flag-USP49CA to check the endogenous USP49, PAX9, and MSX1 proteins. The presented immunoblots are representative of three independent experiments (n = 3). F Reconstitution effect of USP49 on endogenous PAX9 or MSX1 in USP49-depleted cells. Protein expression was analyzed by Western blotting using the indicated antibodies. GAPDH was used as the loading control. The presented immunoblots are representative of three independent experiments (n = 3).

Article Snippet: MATERIALS AND METHODS Plasmids, sgRNAs, and shRNAs A mammalian expression vector encoding Myc-Flag-tagged PAX9 was purchased from OriGene (Cat. no. RC200380, Rockville, USA).

Techniques: Biomarker Discovery, Western Blot, Transfection, shRNA, Expressing, Control

Journal: Journal of dental research

Article Title: Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

doi: 10.1177/0022034517729322

Figure Lengend Snippet: Figure 1. Clinical characteristics of probands with a PAX9 mutation. (A) Intraoral photos and panoramic radiographs of probands with distinct PAX9 mutations. Asterisks represent the position of missing teeth. (B) The prevalence of missing teeth in all PAX9 mutation patients (n = 16). L, lower; U, upper. (C) The prevalence of microdontia in all patients with a PAX9 mutation.

Article Snippet: Construction of PAX9 Expression Vectors and Site-Directed Mutagenesis The human PAX9 complementary DNA (cDNA) (accession number: NM_006194; Origene) was subcloned into the pCMVC-Myc expression vector (Beyotime) between 5′-BamHI and 3′-EcoRI sites to generate the pCMV-PAX9-Myc wild-type plasmid.

Techniques: Mutagenesis

Journal: Journal of dental research

Article Title: Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

doi: 10.1177/0022034517729322

Figure Lengend Snippet: Figure 2. Location and conservation analysis of tooth agenesis associated PAX9 mutations. (A) Distribution of mutations identified in patients with tooth agenesis in the PAX9 protein. Reported mutations are labeled in blue. Black indicates novel PAX9 mutations. (B) Schematic diagram of the PAX9 gene. (C) Conservation analysis of affected amino acids in the PAX9 protein among 9 different vertebrate species.

Article Snippet: Construction of PAX9 Expression Vectors and Site-Directed Mutagenesis The human PAX9 complementary DNA (cDNA) (accession number: NM_006194; Origene) was subcloned into the pCMVC-Myc expression vector (Beyotime) between 5′-BamHI and 3′-EcoRI sites to generate the pCMV-PAX9-Myc wild-type plasmid.

Techniques: Labeling

Journal: Journal of dental research

Article Title: Nine Novel PAX9 Mutations and a Distinct Tooth Agenesis Genotype-Phenotype.

doi: 10.1177/0022034517729322

Figure Lengend Snippet: Figure 3. Functional studies of mutant PAX9 proteins. (A) Expression of wild-type and mutant PAX9 proteins detected by Western blot. (B) Relative messenger RNA (mRNA) expression levels of mutant PAX9 at 12 h posttransfection. (C) After treatment with actinomycin D, PAX9 mRNAs underwent decay. The mRNA stability is presented by the percentage of remaining mRNAs at 4 and 8 h posttreatment. *P < 0.05. (D) Subcellular localization of wild-type and mutant PAX9 proteins assessed by immunofluorescence. (E) The transcriptional activation abilities of PAX9 mutants on the BMP4 promoter assessed by luciferase reporter assay. **P < 0.01. (F) Binding of PAX9 mutants to the paired domain consensus sequence CD19-2 (A-ins) assessed by an electrophoretic mobility shift assay.

Article Snippet: Construction of PAX9 Expression Vectors and Site-Directed Mutagenesis The human PAX9 complementary DNA (cDNA) (accession number: NM_006194; Origene) was subcloned into the pCMVC-Myc expression vector (Beyotime) between 5′-BamHI and 3′-EcoRI sites to generate the pCMV-PAX9-Myc wild-type plasmid.

Techniques: Functional Assay, Mutagenesis, Expressing, Western Blot, Immunofluorescence, Activation Assay, Luciferase, Reporter Assay, Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay