FIGURE 6.

Cross-linked desmosomal cadherins contain trans-interacting molecules. A, mDsg2-HA and mDsg2-FLAG were incorporated into desmosomes. Confluent transfected cells were double-stained with anti-desmoplakin antibody, 11-5F, and anti-HA or anti-FLAG. Co-localization of desmoplakin (DP) with HA-tagged or FLAG-tagged mDsg2 was observed under the confocal microscope. Scale bar, 5 μm. B, whole cell extracts of HaCaT cells with stable expression of pC-mDsg2HA or pC-mDsg2FLAG were Western-blotted (equal loading) and showed approximately equal expression of HA-tagged mDsg2 (mDsg2-HA) or FLAG-tagged mDsg2 (mDsg2-Fg) in the clones selected for the experiment. C and D, detection of trans-interacting cross-linked adducts. Transfected cells were mixed in a ratio of 1:2 of the type to be immunoprecipitated to the type to be Western-blotted after IP and cultured at confluent density. The cultures were cross-linked (1 mm SEGS for 10 min), extracted, and immunoprecipitated with the appropriate anti-tag antibody. The IPs were Western-blotted with the antibody used for IP and with the antibody against the other tag. In each case, the IP antibody recognized both the monomer and the cross-linked adduct, whereas the other antibody recognized only the cross-linked adducts.