Potential Canonical Wnt Pathway Activation in High-Grade Astrocytomas
(a) Gel electrophoresis showing expression of key players of the canonical wnt signalling pathway as assessed by RT-PCR. (b) (upper rows) Representative western blot analysis of 3 independent experiments displaying β-catenin expression in whole cell lysates and corresponding cytoplasmic fractions of high-grade glioma cultures. (lower graph) Quantification of the cytoplasmatic β-catenin fractions shown in (b). Expression levels were normalized to A172. (c) Transcriptional activity of β-catenin in glioma cultures as assessed by TOPFLASH/FOPFLASH reporter gene assay. Ordinate shows x-fold induction of the luciferase reporter gene in comparison to the control vector (FOPFLASH). Horizontal lines mark 1.5-fold and 2-fold reporter induction, respectively. Data indicate mean values of at least 3 independent experiments plus SD. (d) For theAPC truncation test, APC cDNA was amplified in five fragments and subsequently transcribed and translated in vitro. Products were separated by SDS-PAGE, blotted on a PVDF membrane, and stained with streptavidin peroxidase. No truncating mutations were found in any of the glioma cultures. Here, cDNA amplification (a), in vitro transcription (b) and in vitro translation (c) of fragment 3 that contains the mutation cluster region are shown. The colon cancer cell line SW480, which contains a homozygous truncating mutation at codon 1338, served as positive control. The molecular weight of the resulting protein fragment in this cell line is thereby reduced from about 70 kDa (full-length fragment 3) to 32 kDa.
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