Cleavage site of human and mouse Dsg1 by ETs. (a) Coomassie brilliant blue staining of SDS-PAGE of purified hDsg1E (left panel) and its ETA cleavage products (right panel). Open arrowhead shows a closely spaced doublet at the predicted molecular weight of 84 kDa, representing the proprotein and mature protein. Cleavage results in products with approximate molecular weights of 50 kDa (arrow 1), 45 kDa (arrow 2), and 34 kDa (filled arrowhead). (b) Immunoblotting with anti–E-tag antibodies of hDsg1E cleaved by ETA and ETB identifies the 34-kDa band (filled arrowhead) as the carboxy-terminal fragment. Open arrowhead indicates uncleaved hDsg1E. (c) Coomassie blue staining of SDS-PAGE of the carboxy-cleavage fragments of hDsg1E (filled arrowheads) cleaved by ETB or ETD and purified on an anti–E-tag Sepharose column. (d) Peptide sequencing (residues following "1" in light gray) of the 50-kDa cleavage product (arrow 1 in a) showed the amino acid sequence of the amino terminus of hDsg1E lacking the signal peptide (residues in green) but still containing the propeptide (residues in blue). Peptide sequencing (residues following "2" in light gray) of the 45-kDa peptide (arrow 2 in a) identifies the amino terminus of mature hDsg1 (residues in black). (e) Cleavage site in Dsg1. Amino-terminal peptide sequencing (residues in gray) of the 34-kDa carboxy terminus of hDsg1E (filled arrowheads in a and c) and mDsg1E (SDS-PAGE not shown) cleaved by ETs. Peptide sequence in black indicates mouse and human Dsg1 and Dsg3 around the cleavage site. Shading shows peptide identity. AA, amino acid.