FIGURE 4.

Dominant-negative Rab5 inhibits agonist-induced syndecan-1 shedding. NMuMG cells were transduced with or without adenovirus harboring LacZ, GFP-WT Rab5, or GFP-DN Rab5 and cell surface syndecan-1 expression was assessed by: A, mild trypsin digestion and dot immunoblotting of trypsinates (mean ± S.E., n = 4) and B, immunostaining with 281-2 anti-syndecan-1 antibodies and Alexa 594 donkey anti-rat IgG antibodies. C, NMuMG cells were infected with the indicated adenovirus vectors and stimulated with or without PMA (1 μm), ceramide (0.1 mm), or S. aureus β-toxin (5 μg/ml) for 4 h at 37 °C and levels of syndecan-1 ectodomains in the conditioned medium were quantified by dot immunoblotting (mean ± S.E., n = 4). D, A549 cells were transduced with the indicated adenovirus vectors and stimulated with PMA or S. aureus β-toxin. The concentration of syndecan-1 ectodomains in the conditioned medium was measured by dot immunoblotting using the B-B4 anti-human syndecan-1 ectodomain antibody (mean ± S.E., n = 4).