Figure 1.

SNAP23-syntaxin13-VAMP3 interaction in CHO cells is stimulated by adhesion to FN. CHO-K1 cells were held in suspension after pretreatment with 1 mM NEM and 2 mM DTT (Susp), held in suspension after pretreatment with 1 mM NEM (Susp + NEM) or plated on FN (FN) for 20 min. The cells were lysed, and the indicated SNAREs were immunoprecipitated with antibodies coupled to sepharose beads from 1-1.5 mg of cellular protein. 10 μg aliquots of the lysates from which SNAREs were immunoprecipitated were run in 'Lysate' lanes. 'S13 Ab'/'SN23 Ab'/'GS15 Ab' indicate syntaxin13, SNAP23 and GS15 antibody control immunoprecipitations, where antibodies coupled to sepharose beads were incubated with lysis buffer, washed, and analyzed. (A) Syntaxin 13 immunoblot of SNAP23 immunoprecipitates showing immunoprecipitation of syntaxin 13 from cells plated on FN and cells treated with NEM, upper blot, and the same membrane reprobed for SNAP23, lower blot. (B) Syntaxin 13 immunoblot of GS15 immunoprecipitates, upper blot, and the same membrane reprobed for GS15, lower blot. (C) Syntaxin13 immunoblot of VAMP4 immunoprecipitates, and the same membrane reprobed for VAMP4, lower blot. (D) CHO-K1 cells, held in suspension (Susp) or plated on FN (FN), were lysed and syntaxin13 was immunoprecipitated with anti-syntaxin13 antibody. The syntaxin13 immunoprecipitates were washed and an aliquot (10%) of the eluate was analyzed by SDS-PAGE-western blot for syntaxin13, upper blot, and co-precipitated VAMP3, middle blot. The remainder of the syntaxin13-immunoprecipitate was re-suspended and immunoprecipitated with anti-VAMP3 antibody and then analyzed by SDS-PAGE-western blot for SNAP23, lower blot.