FIGURE 8.

Decreased binding of pro-NT to sortilin-4A within cells. A, immunostaining of HEK 293 cells transfected with constructs for full-length (fl)-sortilin-WT or sortilin-4A with an antibody against sortilin. Protein expression levels tested by Western blot analysis of cell lysates. B and C, cells were labeled with ligands at 4 °C followed by endocytosis for 30 min at 37 °C before fixation and internalized ligand visualization by staining. Ligands applied were either a sortilin antibody (against the receptor extracellular domain; α-sortilin), the receptor propeptide (GST-propept) (B), GST-NGFpro, or BDNFpro (C). D, cells co-transfected with constructs for sortilin variants devoid of endocytosis (fl-sortilin-WT^Δendo or fl-sortilin-4A^Δendo) together with p75^NTR were immunoprecipitation using an antibody against p75^NTR. Lysates and precipitated proteins were subjected to SDS-PAGE and analyzed by Western blotting for either p75^NTR or sortilin as indicated.