Fig. 5. Effect of protease inhibitors and MßCD on NTF-NgR release. (A) SH-SY5Y cells expressing NgR were incubated for 3 hours with OptiMEM in the absence (–) or presence of either 10 µM E-64, 10 µM AEBSF, 10 µM Pepstatin, 10 µM PKF226-967 or 10 mM NH₄Cl. NgR in the lysate and medium was detected by western blot analysis with the anti-V5 antibody. (B) Structure of PKF226-967 and table showing IC₅₀ values of this compound for the indicated zinc metalloproteinase family members and the shedding of TNF- from human peripheral blood mononuclear (hPBMC) cells (Kottirsch et al., 2002). (C) Cells were incubated for 3 hours with Opti-MEM in the absence (–) or presence of 10 µg/ml of the indicated recombinant human TIMP. NgR in the lysate and medium was detected by western blot analysis with the anti-V5 antibody. (D) Cells were incubated for 30 minutes with OptiMEM in the absence (–) or presence (+) of 10 mM MßCD with or without 10 µM PKF226-967. NgR in the lysate and medium was detected by western blot analysis with the anti-V5 antibody.