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Fig. 3. K1014chim expression in cultured keratinocytes from newborn mutant mice. The antibodies used [K5, K10, Desmoplakin (Dp)] are indicated. Both keratin antibodies (K5, K10) used in this experiment were raised against the C-terminal tail domains of their targets. The K10 antibody therefore recognizes both K1014chim and K10. However, the keratinocytes shown were cultured for 8 days in low calcium medium to eliminate differentiating cells. Under these culture conditions, K10 is not expressed in wild-type control cells (see Fig. 4). The homogenous staining of mutant cultures with the K10 antibody therefore indicates expression of the K1014chim protein. (a-c) Staining of mutant cells (maintained in low calcium medium) with the keratin antibodies demonstrates co-distribution of K5 and K1014chim, suggesting that the chimeric keratin can form a functional intermediate filament cytoskeleton with its partner K5. (d-f) Keratinocytes cultured in high calcium medium for 1 hour to induce cell junction formation. Dp is a general marker for desmosomes (reviewed in Cheng et al., 2005; Cheng and Koch, 2004). Dp antibodies stain cell-cell borders in a punctuated fashion. Each dot represents a single desmosome. The double staining with Dp and K10 antibodies indicates that the K1014chim filaments terminate in desmosomes at the plasma membrane.
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