rabbit anti mouse rank  (Bioss)

Journal: Cell Death & Disease

Article Title: Oxytocin receptor induces mammary tumorigenesis through prolactin/p-STAT5 pathway

doi: 10.1038/s41419-021-03849-8

Figure Lengend Snippet: A Serum prolactin levels, n = 5. B Serum progesterone levels, n = 5. C Immunoblotting analysis of OXTR, p-STAT5, STAT5, and RANKL from fourth mammary gland of ++ Oxtr , WT, and ++ Oxtr tumors. GAPDH is the loading control. D Immunostaining of p-STAT5 from fourth mammary gland of ++ Oxtr , WT, and ++ Oxtr tumors. Nuclei were stained blue with hematoxylin. Scale bar: 100 μm. E GSEA plots evaluating enrichment of upregulated genes of STAT5-induced mammary tumors (Data from GSE15119 were reanalyzed) in ++ Oxtr tumors. F GSEA plots evaluating enrichment of downregulated genes of STAT5-induced tumors in ++ Oxtr tumors. G Venn diagram displayed the overlap between STAT5-binding genes (Data from GSE74826 were reanalyzed) and the upregulated genes in ++ Oxtr tumors. H Ontology analysis of overlapping genes between OXTR-upregulated and STAT5-binding genes. I ChIP-seq profiles of STAT5 on Erbb2 , Akt1 , and Tgfα in mouse mammary gland – (Data from GSE2492061, GSE74826, and GSE82275 were obtained). Data represented as mean ± SD. ** P < 0.01, *** P < 0.001, calculated using two-tailed unpaired t -test.

Article Snippet: The membranes were incubated with primary antibodies rabbit anti-OXTR (ab181077, 1:5000), rabbit anti-STAT5 (94205 s, 1:1000), rabbit anti-Phosoho-STAT5 (9359 s, 1:1000), goat anti-RANKL (AF462, R&D Systems, 1:2000), and rabbit anti-GAPDH (AP0063, Bioworld, 1:10,000) at 4 °C overnight.

Techniques: Western Blot, Immunostaining, Staining, Binding Assay, ChIP-sequencing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Oxytocin receptor induces mammary tumorigenesis through prolactin/p-STAT5 pathway

doi: 10.1038/s41419-021-03849-8

Figure Lengend Snippet: After E0771 cells transplantation, ++ Oxtr females were treated with a vehicle or 200 ug (1 mg/ml) bromocriptine (Br) for 15 days. A Serum prolactin (PRL) levels of WT, ++ Oxtr , and ++ Oxtr females with Br treatment, n = 8. B Whole-mount staining of fourth mammary glands, Scale bar: 500 μm. C Tumor growth ( n = 10) by tumor volume. D Representative photos of tumors. E Tumor weights, WT ( n = 13), ++ Oxtr ( n = 10), and ++ Oxtr with Br treatment ( n = 12). F p-STAT5 immunostaining of tumors. Nuclei were stained blue with hematoxylin. Scale bar: 100 μm. G Immunoblotting analysis of p-STAT5 of tumors. H Gene expression of Erbb2 , Akt1 , and Tgfα in tumors by qPCR, n = 6. Data were represented as mean ± SD. ** P < 0.01, *** P < 0.001, calculated with one-way analysis of variance (ANOVA).

Article Snippet: The membranes were incubated with primary antibodies rabbit anti-OXTR (ab181077, 1:5000), rabbit anti-STAT5 (94205 s, 1:1000), rabbit anti-Phosoho-STAT5 (9359 s, 1:1000), goat anti-RANKL (AF462, R&D Systems, 1:2000), and rabbit anti-GAPDH (AP0063, Bioworld, 1:10,000) at 4 °C overnight.

Techniques: Transplantation Assay, Staining, Immunostaining, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Oxytocin receptor induces mammary tumorigenesis through prolactin/p-STAT5 pathway

doi: 10.1038/s41419-021-03849-8

Figure Lengend Snippet: OXTR overexpression leads to increased prolactin secretion in ++ Oxtr females. Prolactin induces phosphorylation and nuclear translocation of p-STAT5 to promote transcription of genes responsible for cell proliferation and milk proteins ( Csn2 and Wap ). Excessive proliferation of mammary epithelium induces tumorigenesis.

Article Snippet: The membranes were incubated with primary antibodies rabbit anti-OXTR (ab181077, 1:5000), rabbit anti-STAT5 (94205 s, 1:1000), rabbit anti-Phosoho-STAT5 (9359 s, 1:1000), goat anti-RANKL (AF462, R&D Systems, 1:2000), and rabbit anti-GAPDH (AP0063, Bioworld, 1:10,000) at 4 °C overnight.

Techniques: Over Expression, Translocation Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: RANKL-induced expression of tetraspanin CD9 in lipid raft membrane microdomain is essential for cell fusion during osteoclastogenesis.

doi: 10.1359/jbmr.060308

Figure Lengend Snippet: FIG. 5. (A and B) RANKL-induced expression of CD9 in lipid raft membrane fraction. Sucrose density-gradient isolation of 1% Triton X-100 insoluble lipid raft membrane fraction from RAW264.7 cells cultured in (A) the absence or (B) presence of RANKL (50 ng/ml) for 4 days. 1% Triton X-100-resistant “lipid raft” membrane microdomain is present at fraction 3–5, and “nonraft” membrane and cytosolic fraction are laid in fractions 9–12. The fractions were separated by SDS-PAGE and analyzed by Western blotting with anti-CD9 (a), anti-flotillin-1 (b), anti-RANK (c), and anti-TRAF-6 (d) antibodies. CD9 was detected in nonraft or cytosolic fractions in the control condition, whereas CD9 was distributed when cells were treated by RANKL. These results were consistent with four independent experiments. (C and D) Visualization of lipid raft distribution of CD9. (C) Antibody-induced patching of lipid raft microdomains on RANKL-treated RAW264.7 cell membrane. Alexa598-conjugated cholera toxin B subunit (CT-B) was applied to the cell surface of living RAW264.7 cells, followed by the incubation with anti-CT-B polyclonal antibody, which induces the clustering of different lipid rafts into patches. After fixation, cells were then stained with anti-CD9 antibody (rat) and FITC-conjugated anti-rat IgG antibody. Immunoreactivity for CD9 (a), Alexa598-conjugated CT-B (b), double exposure (c), and Nomarski transmission images (d). CD9 on cell surface and CT-B are largely co-patched (c), suggesting the distribution of CD9 into lipid raft fraction. Scale bar represents 10 m. (D) Immunogold electron microscopy of CD9 and lipid rafts in RAW264.7 cells. Positive colloidal gold particles for CD9 (large particle: diameter 15 nm; small particle: diameter 7 nm) were detected on the cell membrane of RAW264.7 cells, and these deposits were largely co-localized (arrowheads). Scale bar represents 100 nm.

Article Snippet: The samples were separated by SDS-PAGE and transferred to PVDF membranes that were immunoblotted with rat monoclonal anti-mouse CD9 (Clone KMC8; BD PharMingen), mouse monoclonal anti-flotillin-1 (Clone 18; BD Transduction, San Diego, CA, USA), rabbit polyclonal anti-RANK (Clone H-300; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit polyclonal anti-TRAF-6 (Clone H-274; Santa Cruz Biotechnology) antibodies, as described above.

Techniques: Expressing, Membrane, Isolation, Cell Culture, SDS Page, Western Blot, Control, Incubation, Staining, Transmission Assay, Electron Microscopy