Fig. 2. Biochemical characterisation of the cadherin-mediated junctional complex in the T98G and U373MG glioblastoma cell lines. (A) In vitro cell migration assay of T98G and U373MG cells. Serum-free medium containing 0.1% BSA (basal) or conditioned media from the same cells (stimulated) were used as chemoattractants. The data are expressed as the mean number of migrating cells per field±s.d. for triplicate samples from a representative experiment. (B) Western blot analysis of total cadherin, ß-catenin and LIN-7 expression in glioblastoma cell lines grown to confluence. 50 µg aliquots of total glioblastoma cell homogenates were loaded onto 11% SDS-PAGE and immunoblotted with the indicated antibodies. (C) Surface biotinylation assay. After being grown to confluence, the glioblastoma cell lines were surface biotinylated with NHS-ss-biotin. After cell lysis, the surface-biotinylated proteins were recovered using streptavidin beads, loaded onto 10% SDS-PAGE and immunoblotted with the indicated antibodies. The results were densitometrically analysed using the NIH Image 1.61 programme. The corresponding ß-catenin/N-cadherin ratio (expressed as relative units) is shown on the right. (D) Affinity chromatography. Confluent cell lysates were incubated with immobilised GST-LIN-7A fusion protein: the bound material (Bound) was resolved by 10% SDS-PAGE and immunostained for ß-catenin. (E) Co-immunoprecipitation of ß-catenin with the LIN-7 antibody. Lysates of confluent cells were immunoprecipitated with the LIN-7 antibody (IP), and the immunocomplexes were resolved by 11% SDS-PAGE and immunoprobed for ß-catenin. 10% of the total T98G or U373MG lysate (Lys) used in the experiments was probed with the indicated antibody (D-E). Molecular weight standards expressed in kDa are indicated on the left (B-E).

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