Fig 2.

Characterization of NTF tethering. (A) Tethering of NTF by CTF. (Left) E3020X was transfected either alone or cotransfected with a CTF construct that includes a C-terminal Flag sequence and assayed for cleavage as shown. Equal amounts of NTF were present in samples with and without CTF coexpression (Lysate). Nonspecific trapping was excluded by using an irrelevant antibody (α-Myc) for IP. (Right) The immunofluorescence results of HEK cells transfected with GFP-E3020X alone or cotransfected with CTF both intracellular and plasma-membrane. (B) Chemical nature of NTF tethering. Co-IP of NTF by CTF-specific α-Flag from WT-transfected HEK cells was tested under native (N), denaturing and nonreducing (D), or denaturing and reducing (D+R) conditions, as outlined (Right). (C) Concentrated culture medium of cells transfected with each of the constructs was analyzed by Western blotting by using α-LRR (Upper). WT-Flag-IP served as size control for FL and NTF. (Lower) Flag-IP for the corresponding protein lysate probed with α-CT. The intracellular level of E3020X was similar to that of WT when total protein lysates were assayed on Western blot by α-LRR (not shown).