Figure 4.

Calnexin association with CD3 masks their carbohydrate side chains. Calnexin–CD3 complexes were immunoprecipitated from detergent extracts of surface biotinylated BW5147 thymic lymphoma cells using anti-CD3ɛ mAb (145–2C11) either before (Unfrac.) or after the extracts were fractionated using ConA-Sepharose. The population of proteins that did not bind to ConA are referred to as ConA Unbound, whereas proteins that bound to ConA and were eluted using α-methyl α-d-mannoside are referred to as ConA Bound. The resultant immune complexes were incubated in buffer alone (M) or digested with the following glycosidases: jack bean mannosidase (JB), which differentially cuts N-linked oligosaccharides depending upon the presence or absence of terminal glucose residues and endoglycosidase H (EH), which is unaffected by the presence of glucose residues. Digested proteins were blotted to immobilon membranes and the surface-labeled proteins were visualized with HRP-Av. The migration positions of mock-treated or endo H-sensitive (EH^s) CD3γ proteins are indicated. Also indicated are the migration positions of CD3γ proteins that either do contain (glc+) or do not contain (glc−) terminal glucose residues.