Fig. 3. Establishment of intercellular adhesion in homozygous, heterozygous and ß-catenin-null mutant keratinocytes after incubation in medium containing 1.2 mM CaCl₂. (A) Cytoplasmic, TritonX-100-soluble and -insoluble protein fractions were assessed by western blot analysis using the indicated antibodies. Compared are cell equivalents at the ratio of 2:1:1 in the respective fractions. E-cad, PG and ß-cat expression were assessed on the same blot, whereas Pph3, Pph1, -cat and Dsg3 expression were investigated on a second blot. Anti-tubulin and anti-keratin 14 antibodies were used as loading controls on all blots of the cytoplasmic or Triton-insoluble fraction, respectively (here shown for one blot). (B) Double-immunofluorescence staining of cultured mouse keratinocytes from the indicated genotype show linear membrane localization of adherens junction proteins, E-cadherin, -catenin, plakoglobin and insertion of actin filaments in adherens junctions. Similar results were obtained for desmosomal proteins. Experimental procedures and photographic exposures were held constant to obtain semi-quantitative results. (C) Adherens junctions and desmosomes demonstrate identical ultrastructure in wild-type and ß-catenin-null mutant keratinocytes (Scale bars, 435 nm). (D) Intercellular adhesiveness was quantified. Values are means of n=4; bars indicate standard deviations. Note that the differences were not significant. Dsg3, desmoglein3; E-cad, E-cadherin; -cat, -catenin; ß-cat, ß-catenin; PG, plakoglobin; Pph, plakophilin.