Fig. 1. Characterisation of the cadherin-mediated junctional complex in primary cultured cortical astrocytes. (A) Western blot analysis of the total expression of the components of the cadherin-based junctional system in brain, astrocytes and MDCK cells. 20 µg aliquots of homogenates were loaded onto 11% SDS-polyacrylamide gel and immunoblotted with the indicated antibodies. The MDCK cell homogenate was probed with the E-cadherin antibody, and the brain and astrocyte homogenate with the pan-cadherin antibody. (B) Affinity chromatography. The rat cortical astrocyte lysate was incubated with immobilised GST-LIN-7A fusion protein. The bound material (Bound) was resolved on 10% SDS-PAGE and immunostained for ß-catenin. 10% of the total astrocyte lysate (Lys) used in the experiment was probed with the same antibody. (C) Co-immunoprecipitation of ß-catenin with the LIN-7 antibody. The rat cortical astrocytes were extracted in lysis buffer and immunoprecipitated with the LIN-7 antibody (IP: LIN-7) or the pre-immune serum (IP: Pre). The immunoprecipitates were loaded onto 11% SDS-PAGE and immunoblotted with the ß-catenin and LIN-7 antibodies. Molecular weight standards expressed in kDa are indicated on the left (A-C). (D) Confocal analysis of double immunofluorescence staining for LIN-7 and ß-catenin in cultured primary astrocytes. The astrocytes were plated onto poly-L-lysine-coated glass coverslips, cultured until they reached confluence (recently confluent) or had been confluent for a longer period (long confluent) before fixation in 4% paraformaldehyde. Bar, 10 µm.