Fig. 1.

Expression of recombinant HSCF. (A) Schemes of Slit3 constructs: Slit3 full length (amino acids 22–1523) and HSCF (amino acids 1125–1523). Both constructs have a transferrin signal (TS) peptide followed by an HPC4 tag at the N terminus and a 6× His-tag at the C terminus. (B) Slit3 cleavage in vivo. The CMs from N2A cells transfected with full-length (lane 1) and C-terminal (lane 2) hSlit3 containing vector were analyzed by western blot probing with an anti-HPC4 monoclonal antibody. (C) Sequence homology between hSlit2 and hSlit3 around the putative cleavage site (bold letters) identified in hSlit2. (D) MS/MS analysis. Purified HSCF that appeared as a single band on an SDS–PAGE gel was cut out and subjected to MS/MS analysis. Identified peptides are shown in bold letters. (E) SDS–PAGE analysis of purified HSCF in reducing (lane 1) and non-reducing conditions (lane 2).