Anti qki

Cells and tissues were harvested in sampling buffer [62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS)] and heated for 5 minutes at 100°C. Protein concentration was determined with the Bradford assay using a commercial kit purchased from Bio-Rad Laboratories (Hercules, CA, USA). Equal quantities of protein were separated electrophoretically on 10% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were probed with diluted antibody. The expression of target proteins was determined with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (IgG)/anti-mouse IgG (Sigma-Aldrich, St Louis, MO, USA) and enhanced chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturers’ suggested protocols. The membranes were stripped and reprobed with an anti–β-actin mouse monoclonal antibody (Sigma-Aldrich) as a loading control. The related antibodies were anti-Smad2/3, anti–NF-κB inhibitor (IκBα), anti–matrix metalloproteinase 9 (MMP9), anti–phosphorylated (p)-Smad2, anti–p-Smad3, anti-Smad2, anti–vascular endothelial growth factor (VEGF) (Cell Signaling Technology, Beverly, MA, USA), anti-QKI, and anti–green fluorescent protein (GFP) (Abcam, Cambridge, MA, USA).

Glioblastoma cells (5 × 10⁵) were stereotactically implanted into the brains of nude mice (n = 5 per group). The mice were monitored daily and euthanized when moribund. Whole brains were removed, paraffin-embedded, sectioned into 4-μm thick slides, and stained with hematoxylin–eosin (H&E) or with anti-QKI (Abcam), anti-MMP9 (Cell Signaling Technology), or anti-VEGF (Cell Signaling Technology) antibodies. Images were captured using an AxioVision Rel. 4.6 computerized image analysis system (Zeiss, Jena, Germany).