Fig. 2. Characterisation of junctional proteins and keratin content of cell lines used in this study, as well as RNase protection assays of PKP1, Dp, plakoglobin, desmoglein 2 and desmoglein 3 mRNA levels. (A) Lack of PKP1 is associated with reduced levels of the desmosomal components but not E-cadherin, ß-catenin, or keratins. Desmoglein 1, PKP2 and PKP3 are not significantly up- or down-regulated in PKP1 deficient cells. Immunoblotting of total cell extracts from nullpB1, nullpB2, nullPKP1, nullPKP2, normpB2 and normpB2 are shown and labelled nullpB, nullPKP and normpB. The graph beneath represents the levels of protein, determined by densitometry, with respect to the loading control and normalised against normpB protein level (density=1±s.e.m. for 2-3 replica experiments). Pg, plakoglobin; Dscs, pan desmocollin (presence of the single band shown is dependent on resolution of gel used as antibody recognises both isoforms), Dsg1, desmoglein 1; Dsg2, desmoglein 2; Dsg3, desmoglein 3; E-cad, E-cadherin; ß-cat, ß-catenin; PKP2, plakophilin 2; PKP3, plakophilin 3; K14, keratin 14; LP34, antibody reactive to keratins 1, 5, 6, 18. (B) Lack of PKP1 does not affect mRNA expression levels of Dp, plakoglobin, desmoglein 2 or desmoglein 3 when comparing nullpB1 with nullPKP1. RNase protection assay using; (lane 1) yeast tRNA (negative control); (lane 2) PCR fragments used as template to generate probe (positive control); (lane 3) nullpB1 RNA; (lane 4) nullPKP1 RNA; (lane 5) normpB1 RNA; to protect [³²P]dUTP-labelled RNA probes. The graph beneath represents mRNA expression levels relative to GAPDH internal probe and normalised to normpB1 levels (density=1±s.e.m. for 2-3 replica experiments).