Figure 1.

Desmoplakin deletion mutants and their expression in cultured epithelial cells. (Top) Shown are diagrams of truncated mutant desmoplakin proteins whose expression was genetically engineered to be under the control of the SV40 major early promoter and enhancer. In all cases, the FLAG epitope tag was placed in-frame (depicted by the flag). The full-length DP transgene was engineered from part DP cDNA and part DP gene, such that both DPI and DPII would be expressed. The coiled coil rod and flanking globular head and tail segments are demarcated according to the Coils Version 2.1 secondary structure prediction program (www site, ISREC, Switzerland). Numbers shown are in amino acid residues; nomenclature is at right. (Bottom) Western blot analyses of protein extracts from COS epithelial cells transfected with the constructs indicated above and harvested 48 h after transfection. Proteins were resolved by electrophoresis through SDS polyacrylamide gels (percentages noted), transferred to nitrocellulose paper by electroblotting, and then visualized by binding to anti-FLAG. Antibody binding was developed using biotinylated secondary antibodies as outlined by Kouklis et al. (1994). (-control) Extracts from mock-transfected cells. Migration of size standards are indicated at left, in kD. Since some transfections were conducted at different times and since variations existed in plasmid preparations and transfection efficiencies, loadings were done to reflect similar transgene protein levels, rather than total protein levels.