Glycosylation analysis and cell surface protein biotinylation of plexin B3. (A) COS-7 cells were transfected with pIRES/B3 and lysed. Untreated lysate served as positive control (B3 pIRES). To analyze cell surface expression and glycosylation of B3 total cell lysates were treated with deglycosylating enzyme Endo H (EndoH). Alternatively, growing cells were incubated by co-translational glycosylation-inhibitor tunicamycin (Tunicamycin). Non-transfected COS-7 cells served as negative control. Lysates were analyzed by Western blot using B3-specific antibody pAbB3-B. (B) After biotinylation of cell surface proteins of cells transfected with pIRES/B3 and immunoprecipitation with pAbB3-B, B3 located on the cell surface was detected in Western blot with alkaline phosphatase-conjugated streptavidin (Surface biotinylation). Non-transfected cells treated as described before served as negative control (COS-7).