Journal: Scientific Reports

Article Title: FAM20A binds to and regulates FAM20C localization

doi: 10.1038/srep27784

Figure Lengend Snippet: ( A ) Comparison of gene expression among Fam20 family members. Quantitative real-time PCR analysis was performed using 5-week-old mouse tissues (brain, heart, lung, kidney, calvaria, tooth). The mean fold change in the expression of each Fam20 member was calculated based on the normalization to that of glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) using the value of Fam20a in brain as a calibrator. The values are shown as the mean + S.D. based on triplicate assays. The expression of Fam20a is indicated by blue bars, Fam20b by red and Fam20c by green. ( B ) Immunohistochemical analysis of FAM20A and FAM20C in adult mouse incisors. FAM20A (a,d) and FAM20C (b,e) were both localized in the same cell/tissue types. No immunoreactivities were observed when non-immune goat serum was used as a negative control (c,f). Scale bar, 50 μm. Images of odontoblasts and ameloblasts were shown at a higher magnification on the right corner of each image (scale bar, 10 μm). Pu; pulp, Od; odontoblasts, PD; pre-dentin, D; dentin, E; enamel, Am; ameloblasts, Si; stratum intermedium.

Article Snippet: The plasmids containing the full length sequence of human FAM20A and FAM20C were purchased from OriGene and used as PCR templates.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Negative Control

Journal: Scientific Reports

Article Title: FAM20A binds to and regulates FAM20C localization

doi: 10.1038/srep27784

Figure Lengend Snippet: ( A ) Binding assay in a cell culture system. The 293 cells were transiently transfected with FAM20A-Flag (lane 2) and FAM20C-HA (lanes 2 and 3). Cell lysates were prepared and immunoprecipitated (IP) with anti-Flag antibody. The interaction was detected by Western blotting (WB) with anti-HA antibody (upper panel). Presence of FAM20C-HA (middle panel) and FAM20A-Flag (lower panel) in the same lysates was verified. ( B ) FAM20A-FAM20C interaction is independent from FAM20C kinase activity. The 293 cells were transfected with FAM20A WT-HA and FAM20C WT-V5 (WT), FAM20C D478A-V5 (DA), or FAM20C P328S-V5 (PS). Cell lysates were immunoprecipitated with anti-V5 antibody and the interaction was detected by WB analysis with anti-HA antibody (upper panel). Presence of FAM20A WT-HA (middle panel) and FAM20C forms (lower panel) in the same lysates was confirmed. ( C ) Binding assay by GST pull down. The rhFAM20C WT-V5/His or rhFAM20C D478A-V5/His protein was incubated with either GST protein alone or GST-FAM20A protein and coupled to glutathione beads. After washing the beads, the bound proteins were visualized by WB analysis with anti-V5 antibody (upper panel). Presence of rhFAM20C WT-V5/His (upper panel, lane 5), rhFAM20C D478A-V5/His (upper panel, lane 6), GST and GST-FAM20A (lower panels) proteins were assessed by WB analysis.

Article Snippet: The plasmids containing the full length sequence of human FAM20A and FAM20C were purchased from OriGene and used as PCR templates.

Techniques: Binding Assay, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: FAM20A binds to and regulates FAM20C localization

doi: 10.1038/srep27784

Figure Lengend Snippet: The 293 cells were transfected with pc3-FAM20A WT-Flag (WT), pc3-FAM20A R392Pfs21-Flag (R392Pfs21) or pc3-FAM20A G331D-Flag (G331D) together with pc3.1-FAM20C WT-V5/His (FAM20C-V5). Conditioned media (CM) were collected and IP-WB analysis was performed to detect extracellular FAM20C (upper panel). The intracellular expression of FAM20C was verified by WB analysis with cell lysates (Lys, middle panel) and that of FAM20A WT and AI-mutants was also confirmed by IP-WB analysis with cell lysates (lower panel). FAM20C secretion is accelerated with FAM20A WT co-transfection as compared to FAM20C transfection alone (upper panel, lane 2 vs. 1ane 1). AI-associated FAM20A mutants, R392Pfs21 (upper panel, lane 3) and G331D (upper panel, lane 4), fail to enhance the extracellular FAM20C accumulation.

Article Snippet: The plasmids containing the full length sequence of human FAM20A and FAM20C were purchased from OriGene and used as PCR templates.

Techniques: Transfection, Expressing, Cotransfection

Journal: Scientific Reports

Article Title: FAM20A binds to and regulates FAM20C localization

doi: 10.1038/srep27784

Figure Lengend Snippet: ( A ) Conditioned media and cell lysates were collected from Wild-type (WT) and knockout (KO) of Fam20a -KO mouse-derived MEF cells. IP-WB analysis with anti-FAM20C antibody was performed to detect extracellular FAM20C (top panel). Intracellular FAM20C (upper middle panel) and intracellular FAM20A (lower middle panel) were detected by WB analysis with anti-FAM20C and anti-FAM20A antibodies, respectively. The expression of β-TUBULIN was shown (bottom panel) as a loading control. Extracellular FAM20C was not detected in KO MEF cells, although intracellular FAM20C levels were comparable between WT and KO cell cultures. Three independent experiments were performed and the representative set of images were shown. The band intensities of intracellular FAM20C normalized to β-TUBULIN in the same lysate were calculated and the values are expressed as the mean + SD (n = 3) (graph on the right). There was no statistical difference of normalized intracellular FAM20C expression between WT and KO. ( B ) MC3T3-E1 osteoblastic cells were cultured with conditioned media (CM) from Fam20a WT and KO MEF cells, and impact of FAM20A-mediated FAM20C secretion was assessed by in vitro mineralization assay. Mineralized nodule formation was impaired in osteoblasts the presence of CM from Fam20a KO MEF cells. Three independent experiments were performed and the representative set of images were shown. The concentration of Alizarin Red S dye extracted from each cell culture matrix was calculated and the values are expressed as the mean + SD (n = 3) with statistical analysis. The asterisk above the bar graph indicates the presence of statistical difference between CM from WT and CM from KO treatment groups. *p < 0.01.

Article Snippet: The plasmids containing the full length sequence of human FAM20A and FAM20C were purchased from OriGene and used as PCR templates.

Techniques: Knock-Out, Derivative Assay, Expressing, Cell Culture, In Vitro, Mineralization Assay, Concentration Assay

Journal: Scientific Reports

Article Title: FAM20A binds to and regulates FAM20C localization

doi: 10.1038/srep27784

Figure Lengend Snippet: A model for the role of FAM20A in the FAM20A-FAM20C complex. FAM20A binds to FAM20C, in which FAM20C may be allosterically modulated by FAM20A, leading to secretion. In the case of FAM20A with Amelogenesis Imperfecta (AI) mutation or absence of FAM20A (i.e. KO), FAM20C secretion does not occur, which consequently results in poor osteoblast mineralization.

Article Snippet: The plasmids containing the full length sequence of human FAM20A and FAM20C were purchased from OriGene and used as PCR templates.

Techniques: Mutagenesis

Journal: Scientific Reports

Article Title: FAM20A binds to and regulates FAM20C localization

doi: 10.1038/srep27784

Figure Lengend Snippet: Primer sequences.

Article Snippet: The plasmids containing the full length sequence of human FAM20A and FAM20C were purchased from OriGene and used as PCR templates.

Techniques: Sequencing