Journal: Cell Death & Disease
Article Title: Notch1 is a prognostic factor that is distinctly activated in the classical and proneural subtype of glioblastoma and that promotes glioma cell survival via the NF-κB(p65) pathway
Figure Lengend Snippet: Notch1 regulates the NF-κB(p65) pathway a Following transfection of U87, U251, and LN229 cells with shRNA, the expression levels of Notch1, NICD, Hes1, p65, cylinD1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9 were detected by western blotting. β-Tubulin was used as a loading control. b Immunofluorescence staining showed the distribution of NF-κB(p65) in U87, U251, and LN229 cells after shRNA treatment. c Three different cell lysates were denatured and then immunoprecipitated with antibodies targeting either NICD or NF-κB(p65). Both the forward and reverse immunoprecipitation showed that NICD bound to NF-κB(p65). Whole immunoglobulin (IgG) was used as a control antibody in the immunoprecipitation assays
Article Snippet: The primary antibodies used in this study targeted the following proteins: Notch1, Hes1, Nestin, Tuj-1, CD133, and GFAP (Abcam, USA; dilution 1:1000); and NICD, NF-κB(p65), cyclin D1, p21, Bcl-2, pro-caspase-3, cleaved caspase-3, pro-caspase-9 and cleaved caspase-9 antibodies (Cell Signaling Technology (CST), USA; dilution 1:1000). β-Tubulin expression (CST; dilution 1:2000) was used as a loading control to normalize the results.
Techniques: Transfection, shRNA, Expressing, Western Blot, Immunofluorescence, Staining, Immunoprecipitation