FIGURE
Fig. 4
- ID
- ZDB-FIG-220803-30
- Publication
- Cui et al., 2022 - PARD3 gene variation as candidate cause of nonsyndromic cleft palate only
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Fig. 4
Truncated PARD3-c.1012dupG variant changed the localization of the wild-type full-length PARD3 protein. (A) MCF-10A cells stably expressing Flag-tagged PARD3-c.1012dupG or full-length PARD3 were developed by lentiviral infection and puromycin selection and formed apical lumens after 10 days of 3D culture in Matrigel. The cysts were stained for Flag-tagged PARD3 (wild-type or mutant) (green) and the basolateral membrane marker β-catenin (red). The truncated PARD3(c.1012dupG) was mainly localized to the basal compartment, while the full-length PARD3 was mainly localized to the lateral and apical areas. The arrow points to the presence of truncated PARD3 at the basal region. Arrowheads point to the apical region. Scale bar = 10 μm. (B) Mass spectrometry analysis of PARD3-c.1012dupG products identified endogenous full-length PARD3 as the candidate interacting protein. Plasmids expressing SBP-His8-tagged PARD3-c.1012dupG or empty vector were stably transfected into HEK-293T cells, and the cells were harvested and lysed 72 h after selection with hygromycin B. Peptides derived from the trypsin digestion of mutant PARD3 pull down complex were analysed by LC–MS/MS. Herein, we used PARD3338-1273 to refer to the C-terminal signal of endogenous PARD3 bound by the PARD3-c.1012dupG protein. The number of peptide hits for the C-terminal signal of endogenous PARD3 (PARD3338-1273) is shown as a pie chart and table. (C) Endogenous PARD3 interacted with Flag-tagged PARD3-p. E338Gfs*26. Flag-tagged PARD3-p. E338Gfs*26 was immunoprecipitated from the cell lysate of HEK-293T cells stably expressing Flag-tagged PARD3-p. E338Gfs*26, and the coimmunoprecipitation product was analysed by anti-PARD3 (C-terminal immunogen) and anti-Flag immunoblotting. (D) Substantial proportion of endogenous PARD3 colocalized with Flag-tagged PARD3-c.1012dupG mainly at the basement membrane in 3D-cultured MCF-10A cells. MCF-10A cells stably expressing Flag-tagged PARD3-c.1012dupG or empty vector were grown in Matrigel, and endogenous PARD3 was analysed with immunofluorescent staining using C-terminal PARD3 antibody (green). Mutant PARD3 was visualized by Flag antibody (red), and nuclei were stained with DAPI (blue). Scale bar = 7.5 μm
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Expression Data
Expression Detail
Antibody Labeling
Phenotype Data
Phenotype Detail
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Full text @ J. Cell. Mol. Med.