Fig. 1. Expression of Ihh variants in COS-1 and CFK-2 cells. (A) Schematic representation of the cDNAs for Ihh, its myc-tagged variant, and its N-terminal domain (N-Ihh), used by insertion into the pcDNA3 mammalian expression vector. Indicated are the internal signal sequence and the conserved cleavage site between Gly241 and Cys242. (B) Western blot analysis of lysates (lanes 1-4; 9-10) or conditioned media (lanes 5-8; 11-12) from COS-1 cells transiently transfected with pcDNA3 (lanes 1,5,9,11), Ihh (lanes 2,6), Ihh-myc (lanes 3,7,10,12) or N-Ihh (lanes 4,8). The detection of a 46 kDa and 19 kDa species in lysates from Ihh or Ihh-myc transfectants was facilitated by the use of -Ihh-N antibody specific to the N-terminal domain of Ihh (lanes 1-8). The 26 kDa C-terminal domain of Ihh was identified with the use of an anti-myc antibody (lanes 9-12). (C) Western blot analysis of COS-1 cell lysates following transient transfection with pcDNA3, N-Ihh-pcDNA3 or N-Ihh W160G-pcDNA3. The 19 kDa peptide species attributed to the N-terminal domain of Ihh in its normal and mutant forms was detected by the -Ihh-N antibody.

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