PMC:5512188 / 1119-5914 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/5512188","sourcedb":"PMC","sourceid":"5512188","source_url":"https://www.ncbi.nlm.nih.gov/pmc/5512188","text":"1 Data\nSgo1 expression pattern largely agreed between the X-Gal stain and immunostain at both E9.5 and E10.5. In both stages, Sgo1 was expressed robustly in the heart and the neural tube (Fig. 1. A-B’). Closer observation of the neural tube revealed predominant localization of the X-Gal signal to its outer border whereas the immunostain marked the SGO1 protein in both the outer and inner borders (Fig. 1C and D). During the later stages of retinal development in E15.5 and E18.5, the X-gal signal diminished from faint to little signal (Fig. 1E–F′). SGO1 localized to cells in the intestinal mesenchymal layers in E10.5 and E12.5. At E15.5, SGO1 localized to the cells at the border of the developing smooth muscle layer and serosa (Fig. 1G–I).\nFig. 1 Sgo1 expression in developing mice embryo. X-Gal stained sections were counterstained with Nuclear Fast Red and the anti-SGO1 immunostained sections were counterstained with methyl green. (A, B) A transverse section comparison between X-gal stained embyro versus anti-SGO1 immunostained embryo. (B′) A transverse section of E10.5 anti-SGO1 immunostain showed localization in CAC. (C, D) A comparison of Sgo1 expression in the neural tube between X-gal and anti-SGO1 immunostain at corresponding stages. (E) E15.5 retina showed minimal X-gal staining. (F, F′) E18.5 retina showed little X-gal stain. (G–I) Intestinal layer in corresponding stages. Lower aerobar indicates the intestinal epithelia. Upper aerobar indicates the intestinal mesenchyme. SGO1 localized to cells in the intestinal mesenchymal layers in E10.5 and E12.5. At E15.5, SGO1 localized to the cells at the border of the developing smooth muscle layer and serosa [black arrowheads]. LV: left ventricle, RV: right ventricle, OFT: outflow tract, CAC: common atrial chamber.\nX-Gal staining of 4 month old adult mice retina showed no discernable signal (Fig. 2A, A′). Anti-SGO1 immunostaining with 3C11 antibody (Abnova; H00151648-M01) showed SGO1 localization in select population of retinal neurons (Fig. 2B); also, shown with monoclonal antibody against SGO1 [3]. Furthermore, SGO1 localized in intestinal ganglion cells of adult mice colon that co-localized with anti-TUBB3, a marker for post-mitotic neurons (Fig. 2C). SGO1 in the cerebellum preferentially localized to the cells in the white matter region (Fig. 2. D). Intracellularly, we identified cells in the cerebral cortex that distinctly shows anti-SGO1 within the boundary of DAPI signal as well as cells which displayed anti-SGO1 signal outside the region positive for DAPI signal (Fig. 2E).\nFig. 2 Sgo1 expression in 4 month old (mo) adult mice tissues. (A, A′) 4mo retina showed no visibly recognizable X-Gal signal. (B) 4mo retina showed SGO1 protein localization in subset(s) of retinal neuronal cells using anti-SGO1 3C11 antibody. (C) SGO1 co-localized with TUBB3 positive intestinal ganglion cells in mouse colon. (D) SGO1 positive cells were largely localized in the white matter of the cerebellum [white arrowhead]. (E) SGO1 localization to the nuclear region of cells [white arrowhead] as well as cells with mutually exclusive localization of anti-SGO1 and DAPI signal [yellow arrowheads]. (F) 4mo mice testis showed punctate anti-SGO1 signal overlapping with methyl green nuclear stain in cells at the spermatogonium niche [red arrowhead]. (G, G′) Majority of hepatocytes showed nuclear SGO1 localization in normal 4mo wild type mice liver. (H) E15.5 mouse heart showed lack of SGO1 localization at the atrioventricular cushion.\nSpermatogonia, in the adult testis reside in the niche at the border of seminiferous tubule wall [1], displayed punctate anti-SGO1 signal, distinctly overlapping with DNA binding counterstain: methyl green. Hepatocytes in the liver are quiescent under normal conditions [2]. SGO1 localized diffusely throughout the nucleus in nearly all morphologically identifiable hepatocytes.\nNo discernable false positive signals were observed under same experimental conditions in immunostains and X-gal stain (Fig. 3). Western blot of adult mouse tissues against SGO1 protein showed a band in the predicted region (59 kDa) of SGO1 protein size with no discernable unspecific binding (Fig. 3.). Refer to the following Gene Expression Patterns article for results and discussion [3].\nFig. 3 Negative controls for immunohistochemistry and X-Gal stain. Western blot of 4mo mice. (A) No primary antibodies control for anti-SGO1, RBPMS immunofluorescence stain did not show any unspecific signal. (B) No primary antibodies control for anti-SGO1 DAB-immunostain did not show any unspecific signal. (C–E) Wild type organs processed and incubated in X-Gal in the same conditions did not show any positive X-Gal signal. (F) Western blot performed with anti-SGO1 ab58023 on adult mice tissues.","divisions":[{"label":"Title","span":{"begin":3,"end":7}},{"label":"Figure caption","span":{"begin":749,"end":1794}},{"label":"Figure caption","span":{"begin":2576,"end":3523}}],"tracks":[{"project":"2_test","denotations":[{"id":"28748208-21664404-24678030","span":{"begin":3622,"end":3623},"obj":"21664404"},{"id":"28748208-21690373-24678031","span":{"begin":3795,"end":3796},"obj":"21690373"}],"attributes":[{"subj":"28748208-21664404-24678030","pred":"source","obj":"2_test"},{"subj":"28748208-21690373-24678031","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#adec93","default":true}]}]}}