Fig. 1.

Cleavage of syndecan-4 by ADAMTS proteases. (A) 293T cells were co-transfected with syndecan-4 and the indicated protease or a plasmid control. 48 h after transfection, equal amounts of CL were heparinase-digested and resolved by SDS-PAGE. WB analysis was performed with a specific antibody for the cytosolic fragment of syndecan-4 (S4 Cyto Ab). (B) 293T cells were co-transfected with syndecan-4 and ADAMTS1 or an inactive form of ADAMTS1, and evaluated as in A with S4 Cyto Ab (upper panel) or an antibody recognizing ADAMTS1 (lower panel). (C) CL from cells transfected as in B were heparinase-digested, resolved, and WB analysis was then performed with a S4 Cyto and an HA antibody. (D) CM from cells transfected as in B were concentrated and resolved on 16% Tricine gels. WB analysis was performed with an HA antibody. (E) 293T cells were transfected with syndecan-4. For first panel, cells were treated with MCD 24 h after transfection in the absence of serum, and lysed after 24 h of treatment. For second panel, an increasing number of cells (first lane: 2 × 10⁵, second lane: 6 × 10⁵, third lane: 12 × 10⁵) was lysed 48 h after transfection. In all cases, equal amounts of CL were heparinase-digested, resolved, and WB analysis was performed with the S4 Cyto Ab. (F) 293T cells were co-transfected with syndecan-4 and ADAMTS1 as indicated. 24 h after transfection, cells were treated with BB94, PMA, or thrombin in the absence of serum, and lysed after 24 h of treatment. Then, CL were heparinase-digested, resolved, and WB analysis was performed with HA and S4 Cyto Abs. A9, A10, A15, A17: ADAM9, 10, 15 and 17; ATS1, ATS4: ADAMTS1 and 4; MMP7: matrix metalloproteinase 7; MT1, membrane type 1-matrix metalloproteinase; C: control; FL Syn4: full length syndecan-4; Cl Syn4: Cleaved syndecan-4.