Figure 3.

Glycosidase-sensitivity experiment demonstrating the glycosylation state of mShh precursor in normal and sterol-deprived cells. (A) Effects of sterol deprivation on the subcellular localization of mShh and the steady-state partition between precursor and mature forms of mShh. Treatment of samples with EndoH is indicated with an “E” whereas treatment of samples with PNGase F is indicated with a “P”. (B) Effects of sterol deprivation on the subcellular localization of the transferrin receptor (Tf^R). CHO-7 cells, where indicated stably expressing mShh, were grown for 48 h in medium A. Four hours before harvest, the media was replaced, as indicated, with sterol-depriving medium. At harvest, membrane fractions were prepared, denatured, and incubated in the presence or absence of PNGase F or Endo H (see Materials and Methods). The samples were then subjected to SDS/PAGE (9% for mShh; 6.5% for Tf^R control), transferred to Immobilon-P, and immunoblotted with either mouse α-FLAG(M2) or mouse α-Tf^R primary antibodies and donkey α-mouse⋅horseradish peroxidase secondary antibody. Blots were developed by using a chemiluminescent substrate for horseradish peroxidase.