Fig. 8. Phosphorylated GalT I is not expressed on the cell surface. 3T3 fibroblasts were metabolically labeled in either [³⁵S]methionine (a,c) or [³²P]orthophosphate (b), lysed and subjected to anti-GalT I immunoprecipitation. Radiolabeled GalT I is readily detectable from both ³⁵S- and ³²P-labeled cells. Immunoprecipitation using either antibodies against the GalT I catalytic domain (-GalT I) or against a peptide unique to the long cytoplasmic domain (-long GalT I) produced similar results. Immunoprecipitation of Gal T I was inhibited by inclusion of the immunizing peptide (+ peptide). Surface-associated GalT I was determined by immunoprecipitating GalT I from cell surface fractions isolated by biotinylation of intact cells, application to streptavidin agarose, and elution of biotinylated material with ß-mercaptoethanol (BME released). Aliquots of the unbound and BME-released surface fraction are shown. Approximately 7% of the total cellular ³⁵S-GalT I was present in the cell surface fractions (determined by quantitative enzyme assay), whereas none of the phosphorylated GalT I could be found on the cell surface. The arrowheads denote the relative migration of GalT I. Molecular weight markers are shown to the left of each panel.

Return to article