Figure 1.

Inhibition of mShh autoprocessing by sterol deprivation. (A) Schematic representation of the precursor and mature forms of recombinant mShh protein. FLAG indicates the double copy of the FLAG-epitope tag. The inverted filled triangle indicates the site of autoproteolytic cleavage, and the filled oval indicates the site of concomitant attachment of cholesterol. The fork indicates a site for N-linked glycosylation. (B) Pulse-chase experiment demonstrating that sterol deprivation inhibits mShh processing and mShh remains cell associated. CHO-7 cells, where indicated stably expressing mShh, were grown overnight in medium A. One hour before metabolic labeling, the medium was replaced, as indicated, with sterol-depriving medium (see Materials and Methods). Cells were subjected to a 30-min pulse labeling with ³⁵S-labeled Met and 30-min chase. Recombinant mShh was immunoprecipitated from detergent lysates or media with mouse α-FLAG(M2)⋅agarose and subjected to SDS/PAGE (12% gel).