Fig. 4.

Inhibition of MUC1 cleavage by TACE/ADAM17 inhibits accumulation of MUC1 CTF15. A diagram of C-terminal subunit cleavage by TACE/ADAM17 is presented in panel A. Within the ectodomain amino acid sequence of C-terminal subunit the site of sheddase cleavage is indicated by an arrow and the position of the N-glycosylation site underlined. Cleavage by TACE/ADAM17 would produce CTF15 with an ectodomain “stub” of 27 amino acids and an intact TMD and CTD. After 48 h of serum withdrawal, HES cells were cultured 39 h in serum-free medium containing 1 µM L685458, with or without cytokine stimulation and 50 µM TAPI as indicated (panel B). Shed/secreted protein was probed with antibody 214D4 to quantify MUC1 ectodomain shedding. The immunoblot of cell-associated proteins was probed with antibody CT1 to quantify accumulation of the CTF15. Inclusion of TAPI produced a similar partial reduction in both MUC1 shedding and CTF15 accumulation. Densitometric analysis of the relative contribution of CTF17 and CTF15 to total C-terminal forms demonstrates the reduction of CTF15 accumulation in response to TAPI inclusion. Note the detection of the 8–10 kDa products in lanes from cells treated with cytokines and L685,458 in the absence of TAPI. In panel C, densitometric analyses and ratios of CTF15 signal to 214D4 signal for each indicated treatment in Panel B confirms that the reduction is proportionate. The bars represent the means ±s.d. of triplicate determinations in each case.