Fig. 4. Proteolytic processing of DLL1 in reserve cells. (A) Schematic diagram of the sequential cleavage of DLL1. The full-length DLL1 (DLL1^FL, 90 kDa) is cleaved by an ADAM. The transmembrane and intracellular domain fragment (DLL1^TMIC, 29 kDa) is then cleaved by -secretase and the intracellular domain (DLL1^IC, 26 kDa) is released. The -secretase-mediated cleavage is inhibited by DAPT, the antibody recognition site is located in the intracellular domain of DLL1. (B) C2C12 cells incubated in differentiation medium for 3 days were separated into myotubes and reserve cells, as in Fig. 1, lyzed, and immunoblotted with anti-DLL1 antibody (left panel). Arrows indicate DLL1^FL, DLL1^TMIC and DLL1^IC, respectively; asterisk indicates a nonspecific band (left panels). The position of DLL1^FL corresponds to the position of the radioactive 90 kDa DLL1^FL band detected in the immunoprecipitate from [³⁵S]-labeled C2C12 cells (top right panel). DLL1^TMIC and DLL1^IC contain 10 times less cysteine and methionine residues than the full-length DLL1 and give weak signals in autoradiograms. The identities of DLL1^TMIC and DLL1^IC are confirmed by the relative increase in the abundance of DLL1^TMIC and decrease in the abundance of DLL1^IC after treatment of C2C12 cells with DAPT (bottom right panel).