pitx3 primary antibody  (Bioss)

Journal: Molecular Medicine Reports

Article Title: PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population

doi: 10.3892/mmr.2019.9989

Figure Lengend Snippet: Pedigrees and sequence electropherograms of PITX3 mutations in 3 Chinese families with autosomal dominant congenital cataract. (A) The slit lamp photo of the right eye of the proband in Family 10003. Opacities were exhibited predominantly in the posterior capsule. (B) Pedigrees and sequence electropherograms of PITX3 mutations identified in 3 Chinese CC families. Family 10003 is a four-generation family with PSC. Families 10094 and 10178 are three-generation CC families. Individuals who underwent whole-exome sequencing are marked with triangle and individuals with available DNA samples were marked with asterisk. Probands are indicated by arrow in each pedigree. The black filled shapes signify individuals with cataract conditions, and the oblique line denotes the individuals who have succumbed. The question mark represents the individual identified to carry an asymptomatic mutation. Vertical arrows indicate the mutations in the index patients, respectively. (C) Schematic representation of PITX3 WT and mutated proteins. The green box represents the homeodomain of 60 amino acids and the blue box is the OAR domain of 14 amino acids. The aberrant protein segments caused by mutations are highlighted by the red box. CC, congenital cataract; M, mutant allele; +/WT, wild-type allele; OAR, homeobox protein orthopedia, Aristaless related homeobox and Retinal homeobox protein Rx domain.

Article Snippet: The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C.

Techniques: Sequencing, Mutagenesis

Journal: Molecular Medicine Reports

Article Title: PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population

doi: 10.3892/mmr.2019.9989

Figure Lengend Snippet: Functional analysis of the expression of PITX3 WT and mutants including. Subcellular localization and transactivation activity. (A) Subcellular localization of the PITX3 WT and mutant proteins transfected in HeLa cells. Cells were stained with PITX3 (N-20) primary antibody and Alexa Fluor 568 donkey anti-mouse IgG as a secondary antibody (red); DAPI was used as a nuclear counterstain (blue). Red fluorescence was not observed in HeLa cells without exogenous gene introduction. For cells transfected with PITX3 wild-type and mutants plasmids, the red fluorescence was localized predominantly in the nucleus. Western blot analysis indicated that the protein expression of PITX3 mutants was not affected. (B) Luciferase assay results for PITX3 WT and mutants co-transfected with the (C) pGL3-MIP (+58/-598), (D) pGL3-FOXE3 (−2988/-3722) or (E) pGL3-LEMD2 (−77/-985) reporters in 293T cells. All luciferase activities were normalized to β-galactosidase activity. In comparison with the empty vector pcDNA3.1, the values are indicated as fold changes of luciferase activity. *P≤0.05, **P≤0.01 and ***P≤0.001. PITX3, paired like homeodomain 3; WT, wild-type; MIP, lens fiber major intrinsic protein; FOXE3, forkhead box protein E3; LEMD2, LEM domain-containing protein 2; ns, not significant.

Article Snippet: The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C.

Techniques: Functional Assay, Expressing, Activity Assay, Mutagenesis, Transfection, Staining, Fluorescence, Western Blot, Luciferase, Comparison, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: PITX3 mutations associated with autosomal dominant congenital cataract in the Chinese population

doi: 10.3892/mmr.2019.9989

Figure Lengend Snippet: Summary of 9 different mutations in PITX3 associated with cataract.

Article Snippet: The cells were washed with PBS 3 times, fixed with purity methanol (100% methanol) at −20°C for 10 min, permeabilized with 0.25% Triton X-100 at 25°C for 10 min, blocked with 1% bovine serum albumin (Sangon Biotech Co., Ltd., Shanghai, China) at 25°C for 1 h, and incubated with a primary antibody PITX3 (N-20) (cat. no. sc-19307; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a 1:1,000 dilution at 25°C for 1 h. Following washing 3 times with PBS, the cells were subsequently incubated with secondary antibody Alexa Fluor 594 Donkey anti-Goat IgG at a 1:200 dilution (cat. no. A11058; Thermo Fisher Scientific, Inc.) for 1 h at 25°C.

Techniques: