Human/Mouse Kilon/NEGR1 Antibody

Detection of Human and Mouse Kilon/NEGR1 by Western Blot. Western blot shows lysates of human brain cortex and mouse brain tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human/Mouse Kilon/NEGR1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5394) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). A specific band was detected for Kilon/NEGR1 at approximately 46 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

Detection of Kilon/NEGR1 by Western Blot Negr1 overexpression halts neuroblastoma growth in vitro and in vivo. (A) Western blot analysis of human neuroblastoma (SH5Y), glioblastoma (U87), murine neuroblastoma (N2a), and murine primary cortical neurons. Samples were stained with anti-Negr1 and anti-actin antibodies. (B) We generated stable N2a clones overexpressing Negr1. We validated Negr1 expression by Western blotting. (C) We analyzed the proliferation rate of wild-type N2a cells and of two different clones stably expressing Negr1 (colonies 3 and 11). N2a clones were seeded at a fixed amount (2 × 103 cells/cm2) at day 0. At DIV5, we counted cell number; n = 12, ** p < 0.01 vs. wild-type. (D) We seeded 2 × 103 cells/cm2 N2a wild-type or stably expressing Negr1 (col3 and 11) cells in soft-agar and cultured them for 14 days. Next, we stained cells with crystal violet. (E) The graph reports the number of colonies (cluster encompassing more than 50 cells); n = 6, ** p < 0.01 vs. wild-type. (F) We subcutaneously injected 1 million N2a cells (wild-type, clone 3, and clone 11) in CD1 immunodeficient nude mice. We monitored tumor growth on days 3, 5, 7, 9, 13, and 15 after injection. The image shows representative tumor masses, scale bar = 1 cm. (G) The graph reports tumor volume; n = 6, ** p < 0.01 vs. wild-type. (H) We analyzed tumors by biochemical means. (I) The graph reports N-MYC levels, normalized over S6RP amount, and expressed as fold over wild-type derived tumors (ctrl), ** p < 0.01, *** p < 0.001 vs. wild-type. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37765276), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Kilon/NEGR1 by Western Blot Negr1 overexpression halts neuroblastoma growth in vitro and in vivo. (A) Western blot analysis of human neuroblastoma (SH5Y), glioblastoma (U87), murine neuroblastoma (N2a), and murine primary cortical neurons. Samples were stained with anti-Negr1 and anti-actin antibodies. (B) We generated stable N2a clones overexpressing Negr1. We validated Negr1 expression by Western blotting. (C) We analyzed the proliferation rate of wild-type N2a cells and of two different clones stably expressing Negr1 (colonies 3 and 11). N2a clones were seeded at a fixed amount (2 × 103 cells/cm2) at day 0. At DIV5, we counted cell number; n = 12, ** p < 0.01 vs. wild-type. (D) We seeded 2 × 103 cells/cm2 N2a wild-type or stably expressing Negr1 (col3 and 11) cells in soft-agar and cultured them for 14 days. Next, we stained cells with crystal violet. (E) The graph reports the number of colonies (cluster encompassing more than 50 cells); n = 6, ** p < 0.01 vs. wild-type. (F) We subcutaneously injected 1 million N2a cells (wild-type, clone 3, and clone 11) in CD1 immunodeficient nude mice. We monitored tumor growth on days 3, 5, 7, 9, 13, and 15 after injection. The image shows representative tumor masses, scale bar = 1 cm. (G) The graph reports tumor volume; n = 6, ** p < 0.01 vs. wild-type. (H) We analyzed tumors by biochemical means. (I) The graph reports N-MYC levels, normalized over S6RP amount, and expressed as fold over wild-type derived tumors (ctrl), ** p < 0.01, *** p < 0.001 vs. wild-type. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37765276), licensed under a CC-BY license. Not internally tested by R&D Systems.