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: An aliquot of the GST-FAM20A protein generated was subjected to WB analysis with anti-FAM20A or anti-GST (goat polyclonal, GE Healthcare Life Sciences, product code: 27-4577-01) antibody and the immunoreactivity of the protein was confirmed.

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: Primer sequences.

Article Snippet: An aliquot of the GST-FAM20A protein generated was subjected to WB analysis with anti-FAM20A or anti-GST (goat polyclonal, GE Healthcare Life Sciences, product code: 27-4577-01) antibody and the immunoreactivity of the protein was confirmed.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: FAM20A is a golgi-localized Type II transmembrane protein

doi: 10.1038/s41598-024-57007-z

Figure Lengend Snippet: Membrane localization of FAM20A. ( A ) FAM20A present in the membrane fraction of the expressing cells. Western-blotting analysis of FAM20A in the membrane and soluble fractions extracted from HEK293 cells transiently transfected with the construct expressing FAM20A-HIS. The blot was first probed with an anti-HIS antibody against FAM20A-HIS. The blot was then sequentially probed with an anti-GAPDH and anti-sodium potassium (Na + K + ) ATPase antibody. GAPDH and sodium potassium ATPase were used as a cytosolic protein marker and plasma membrane protein marker, respectively. L, total cell lysate; M, membrane fraction; and S, soluble fraction. ( B ) FAM20A is not secreted out the expressing cells. HEK293 cells were transiently transfected with pCDNA3 empty vector (Vector) or a construct expressing FAM20A-FLAG and/or FAM20C-FLAG. The total cell lysates and conditioned media were harvested and analyzed by Western-blotting with a mouse monoclonal anti-FLAG M2 antibody, and the blot was then stripped and probed with a mouse monoclonal β-actin antibody. The amount of DNA transfected for each construct are indicated. For lanes 1, 2 and 3, 60 µg of total cell lysates were loaded. For lanes 5 and 6, 1 ml of conditioned medium was loaded after concentration; and for lanes 7 and 8, 20 µl of conditioned medium was loaded. Note that β-actin was detectable in lanes 5 and 6 loaded with concentrated medium. The original blots are provided in Supplementary Fig. .

Article Snippet: The primary antibodies used in this study included mouse-monoclonal anti-FLAG M2 antibody (1:1000; Sigma, USA), rabbit anti-FAM20A antibody (1:250; Cat. No: A8496; ABclonal), rabbit-anti-GM130 antibody (1:1000; Cat. No. ab52649; Abcam, USA), mouse monoclonal anti-GM130 antibody (1:1000; Cat. No.: NBP3-23353; Novus Biological, USA), and rabbit-polyclonal anti-Calnexin antibody (1:1000; Cat. No. ab22595; Abcam, USA).

Techniques: Membrane, Expressing, Western Blot, Transfection, Construct, Marker, Plasmid Preparation, Concentration Assay

Journal: Scientific Reports

Article Title: FAM20A is a golgi-localized Type II transmembrane protein

doi: 10.1038/s41598-024-57007-z

Figure Lengend Snippet: Subcellular localization of exogenous FAM20A in 17IIA11 and LS8 cells. 17IIA11 odontoblast-like cells ( A ) and LS8 ameloblast-like cells ( B ) were transiently transfected with the FAM20A-FLAG construct, and were then permeabilized with 0.1% Triton X-100 and immunofluorescently labeled with a mouse monoclonal anti-FLAG M2 antibody that detects FAM20A-FLAG (signal in red) and a rabbit anti-GM130 antibody (signal in green). Nuclei were stained with DAPI (blue). GM130 is a marker for the cis-Golgi network. The merged images show co-localization (yellow) of FAM20A-FLAG and GM130. *Marks the non-transfected cells. Scale bars: 10 µm.

Article Snippet: The primary antibodies used in this study included mouse-monoclonal anti-FLAG M2 antibody (1:1000; Sigma, USA), rabbit anti-FAM20A antibody (1:250; Cat. No: A8496; ABclonal), rabbit-anti-GM130 antibody (1:1000; Cat. No. ab52649; Abcam, USA), mouse monoclonal anti-GM130 antibody (1:1000; Cat. No.: NBP3-23353; Novus Biological, USA), and rabbit-polyclonal anti-Calnexin antibody (1:1000; Cat. No. ab22595; Abcam, USA).

Techniques: Transfection, Construct, Labeling, Staining, Marker

Journal: Scientific Reports

Article Title: FAM20A is a golgi-localized Type II transmembrane protein

doi: 10.1038/s41598-024-57007-z

Figure Lengend Snippet: Subcellular localization of endogenous FAM20A in 17IIA 11 and LS8 cells. Non-transfected 17IIA11 cells ( A ) and LS8 cells ( B ) were permeabilized with 0.1% Triton X-100 and immunofluorescently labeled with a rabbit anti-FAM20A antibody that detects endogenous FAM20A (signal in red) and a mouse monoclonal anti-GM130 antibody (signal in green). Nuclei were stained with DAPI (blue). GM130 is a marker for the cis-Golgi network. The merged images show co-localization (yellow) of FAM20A and GM130. Scale bars: 25 µm.

Article Snippet: The primary antibodies used in this study included mouse-monoclonal anti-FLAG M2 antibody (1:1000; Sigma, USA), rabbit anti-FAM20A antibody (1:250; Cat. No: A8496; ABclonal), rabbit-anti-GM130 antibody (1:1000; Cat. No. ab52649; Abcam, USA), mouse monoclonal anti-GM130 antibody (1:1000; Cat. No.: NBP3-23353; Novus Biological, USA), and rabbit-polyclonal anti-Calnexin antibody (1:1000; Cat. No. ab22595; Abcam, USA).

Techniques: Transfection, Labeling, Staining, Marker

Journal: Scientific Reports

Article Title: FAM20A is a golgi-localized Type II transmembrane protein

doi: 10.1038/s41598-024-57007-z

Figure Lengend Snippet: Subcellular localization of FAM20A in odontoblasts and ameloblasts in mice. Shown are representative images of immunohistochemical results of a sagittally-sectioned mandibular first molar of 4-day-old wild-type mice. The tissue section was immunofluorescently labeled with a rabbit polyclonal anti-FAM20A antibody that detects endogenous FAM20A (signal in red) ( A , D ) and a mouse monoclonal anti-GM130 antibody (signal in green) ( B , E ). Nuclei were stained with DAPI (blue) ( C , F ). GM130 is a marker for the cis-Golgi network. The merged image shows co-localization (yellow) of FAM20A and GM130 in odontoblasts and ameloblasts ( C , F ). ( D – F ) are the higher magnification views of the boxed area in ( A – C) , respectively. od, odontoblasts; am, ameloblasts; and dp, dental pulp. Scale bars: 25 µm.

Article Snippet: The primary antibodies used in this study included mouse-monoclonal anti-FLAG M2 antibody (1:1000; Sigma, USA), rabbit anti-FAM20A antibody (1:250; Cat. No: A8496; ABclonal), rabbit-anti-GM130 antibody (1:1000; Cat. No. ab52649; Abcam, USA), mouse monoclonal anti-GM130 antibody (1:1000; Cat. No.: NBP3-23353; Novus Biological, USA), and rabbit-polyclonal anti-Calnexin antibody (1:1000; Cat. No. ab22595; Abcam, USA).

Techniques: Immunohistochemical staining, Labeling, Staining, Marker

Journal: Frontiers in Immunology

Article Title: FAM20A : a potential diagnostic biomarker for lung squamous cell carcinoma

doi: 10.3389/fimmu.2024.1424197

Figure Lengend Snippet: Expression Levels of FAM20A and FAM20C in LUSC. (A) FAM20A expression across various cancers, as sourced from the GTEx database. (B) FAM20C expression across different cancers, derived from the GTEx database. (C) FAM20A expression in 33 cancer types. (D) FAM20C expression in 33 cancer types. (E) Comparison of FAM20A expression in tumor versus normal samples. (F) Differential expression of FAM20C in tumor samples. (G) Box plots comparing FAM20A , FAM20B , and FAM20C expression in patients with and without LUSC. (H) Expression differences of FAM20A , FAM20B , and FAM20C between LUSC samples and matched non-cancerous samples. (I) ROC curve analysis of FAM20A , FAM20B , and FAM20C in normal versus LUSC samples from the TCGA database. (J) qRT-PCR analysis for FAM20A and FAM20C . (K) Western blot results for FAM20A and FAM20C. (L) Immunohistochemistry (IHC) analysis of FAM20A and FAM20C. Significance levels indicated as * P <0.05, ** P <0.01, *** P <0.001. LUSC, Lung squamous cell carcinoma; Fam20A, Family with Sequence Similarity 20, Member A; FAM20B, Family with Sequence Similarity 20, Member B; FAM20C, Family with Sequence Similarity 20, Member C; ACC, Adrenocortical carcinoma; BLCA, Bladder urothelial carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, Cholangiocarcinoma; COAD, Colon adenocarcinoma; DLBC, Lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, Esophageal carcinoma; GBM, Glioblastoma multiforme; HNSC, Head and neck squamous cell carcinoma; KICH, Kidney chromophobe; KIRC, Kidney renal clear cell carcinoma; KIRP, Kidney renal papillary cell carcinoma; LAML, Acute myeloid leukemia; LGG, Brain lower grade glioma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; MESO, Mesothelioma; OV, Ovarian serous cystadenocarcinoma; PAAD, Pancreatic adenocarcinoma; PCPG, Pheochromocytoma and paraganglioma; PRAD, Prostate adenocarcinoma; READ, Rectum adenocarcinoma; SARC, Sarcoma; SKCM, Skin cutaneous melanoma; STAD, Stomach adenocarcinoma; TGCT, Testicular germ cell tumors; THCA, Thyroid carcinoma; THYM, Thymoma; UCEC, Uterine corpus endometrial carcinoma; UCS, Uterine carcinosarcoma; UVM, Uveal melanoma. ns: not significant difference.

Article Snippet: After blocking with 5% BSA, the membrane was incubated with polyclonal anti-FAM20A (1:1000, Cat. No. 25258–1-AP, Proteintech, USA), and FAM20C antibody (1:1,000, Cat. No. 25395–1-AP, Proteintech, USA) in a primary antibody dilution buffer (Epizyme, China).

Techniques: Expressing, Derivative Assay, Comparison, Quantitative Proteomics, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Sequencing

Journal: Frontiers in Immunology

Article Title: FAM20A : a potential diagnostic biomarker for lung squamous cell carcinoma

doi: 10.3389/fimmu.2024.1424197

Figure Lengend Snippet: Survival Analysis in LUSC Based on FAM20A and FAM20C. Utilizing the GEPIA2 tool, overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in LUSC were analyzed in relation to the expression of FAM20A and FAM20C, with survival plots provided. Significance levels indicated as * P <0.05, ** P <0.01, *** P <0.001. LUSC, Lung squamous cell carcinoma; FAM20A, Family with Sequence Similarity 20, Member A; FAM20C, Family with Sequence Similarity 20, Member C; GEPIA2, Gene Expression Profiling Interactive Analysis 2; OS, Overall survival; DSS, Disease-specific survival; PFI, Progression-free interval.

Article Snippet: After blocking with 5% BSA, the membrane was incubated with polyclonal anti-FAM20A (1:1000, Cat. No. 25258–1-AP, Proteintech, USA), and FAM20C antibody (1:1,000, Cat. No. 25395–1-AP, Proteintech, USA) in a primary antibody dilution buffer (Epizyme, China).

Techniques: Expressing, Sequencing, Gene Expression

Journal: Frontiers in Immunology

Article Title: FAM20A : a potential diagnostic biomarker for lung squamous cell carcinoma

doi: 10.3389/fimmu.2024.1424197

Figure Lengend Snippet: Association of FAM20A and FAM20C levels with Clinical Pathological Traits in LUSC. (A) Overall survival, (B) Disease specific survival and (C) Progress Free interval of FAM20A mRNA levels. (D) Overall survival, (E) Disease specific survival and (F) Progress Free interval of FAM20C mRNA levels. Significant levels indicated as * P <0.05, ** P <0.01, *** P <0.001. LUSC, Lung squamous cell carcinoma; Fam20A, Family with Squance Similarity 20, member A; Fam20C, Family with Squance Similarity 20, member C.

Article Snippet: After blocking with 5% BSA, the membrane was incubated with polyclonal anti-FAM20A (1:1000, Cat. No. 25258–1-AP, Proteintech, USA), and FAM20C antibody (1:1,000, Cat. No. 25395–1-AP, Proteintech, USA) in a primary antibody dilution buffer (Epizyme, China).

Techniques: