Figure 5. ADAM17-mediated p75 ectodomain shedding in NG108-15 cells.

(A) Endogenous expression of Lingo-1, p75, and NgR1 in the NG108-15 cell line before and after differentiation. The NG108-15 cells were differentiated with 1 mM dibutyl cAMP, which resulted in enhanced expressions of NgR1, Lingo-1, and p75. The cells were harvested, lysed, and subjected to Western blots analysis. For NogoR1, we detected minor higher molecular weight bands. This could be because of the association of membranous glycolipids, such as gangliosides, with NogoR1 in cell lysates of NG105-15, that slow the migration of NogoR1 on SDS–PAGE, or may be because of differently glycosylated species. The main 60 kD band represents the NgR1 receptor ectodomain. (B) PMA induced shedding of p75 in the presence or absence of the myelin inhibitor MAG. Briefly, 1 × 10⁵ NG108-15 cells were differentiated by dibutyl cAMP (1 mM) for 3–4 d in six-well cell-culture plates (Nunc). The cells were transferred to conditioned media without FBS and allowed to grow for 24 h. PMA (25 ng/ml) was added to the media and incubated for either 15′ or 30′. Finally, the myelin inhibitor MAG (20 μg/ml) was added followed by an additional 30′ incubation. The culture supernatants (media and secreted proteins) were then harvested, concentrated, and subjected to Western Blot analysis using the p75 specific mAb 8J2. Likewise, the cells were lysed and blotted for the loading control, GAPDH. The results show that PMA augments the shedding of p75 in the presence of the exogenously added myelin inhibitor, MAG. (C) The ADAM17 inhibitor TAPI-1 causes significant inhibition of p75 shedding in absence of PMA. Differentiated NG108-15 cells, in absence of PMA, were incubated for 30′ with 1 mM TAPI-1 (30) before the addition of MAG. MAG was then added, followed by another 30′ of incubation. The supernatants were harvested and processed as before.