Figure 3

Expression of syndecan-1 in GM00701 cells. (A) GM00701 cells were transfected with pMH alone (lane 1) or pMH/Syn-1-HA (lane 2). Forty eight hours after transfection, total protein was extracted from cells with 1% Triton X-100 and equal amounts of protein (20 μg/lane) were separated by 7.5% SDS-PAGE, transferred to PVDF membrane and immunoblotted with anti-Syn-1 polyclonal antisera (1:1000) that was raised against a recombinant his-tagged fusion protein representing full length human Syn-1. Chemiluminescence detection was used to visualize bound antibodies. (B) GM00701 cells were transfected with pMH/Syn-1-HA and cells were selected that stably express Syn-1 (GM00701/Syn-1-HA). Cells were incubated with either TBS (lane 1), 8 M urea (lane 2), or 1% Triton X-100 (lane 3). Insoluble material was pelleted by centrifugation and soluble proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). (C) GM00701/Syn-1-HA cells were incubated without (lane 1) or with (lane 2) heparinase (1 unit/ml) for 5 h at 37°C. Total cellular proteins were extracted with 8 M urea, separated by 6% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). Asterisks indicate the 77 kD, non-glycosylated Syn-1 core protein [36, 37].