Fig. 3. Biochemical evidence that TACE and SAP97 interact in mammalian cells. (A,B) Interaction of endogenous TACE and SAP97. COS-7 cells lysate (lanes 1) was submitted to a mock immunoprecipitation (lanes 2) or to an immunoprecipitation using an anti-SAP97 antibody (lanes 3) as described in Materials and Methods. (A) Results were analyzed by immunoblotting under reducing conditions using the C-terminus-specific TACE antibody. The position of the immature (around 120 kDa) and mature (around 90 kDa) form of TACE is indicated by i and m, respectively. (B) Results were analyzed by immunoblotting under non-reducing conditions using the ectodomain-specific TACE antibody. The position of the mature form of TACE (around 80 kDa) is indicated by m; the arrow head indicates the position of the IgG. (C) PDZ3 domain of SAP97 interacts with immature and mature forms of TACE. Left: Lysates from cells transfected with GFP-SAP97 (lane 1) or GFP-SAP97_DPDZ3 (lane 2) were submitted to immunoprecipitation using a anti-GFP antibody as described in Materials and Methods (lanes 3 and 4, respectively). Results were analyzed by immunoblotting using the C-terminal-specific TACE antibody. Right: Western blot on COS-7 cells lysates using anti-SAP97 antibodies attests for the expression of SAP97 and SAP97_DPDZ3. (D) The PDZ-binding motif of TACE interacts with SAP97. Left: lysates from mock-transfected COS-7 cells (lane 1). Lysates from cells co-transfected with GFP-SAP97 and haTACE (lane 2) or with GFP-SAP97 and haTACEm (lane 3) were submitted to immunoprecipitation using anti-HA antibody as described in Materials and Methods (lanes 4 and 5, respectively). Results were analyzed by immunoblotting using an anti-SAP97 antibody. Right: Western blot of COS-7 cells lysates using anti-HA epitope antibody demonstrates the expression of haTACE and haTACEm. Overexpressed forms of TACE are schematically detailed: black (HA), hemagglutinin epitope; white, disintegrin-like domain (beginning at amino acid 475); gray (mb), transmembrane region; striped, cytoplasmic domain with mutations indicated.

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