Fig. 3. TI-VAMP and cellubrevin interacted specifically in the ER with ectopic syntaxin 1. (A) HeLa cells were either co-transfected with GFP-Cb and Stx1 (1), or transfected with each cDNA alone (2,3), as indicated. 24 hours after transfection, cells were lysed and the extract from co-transfected cells was immediately immunoprecipitated (1), whereas extracts from cells transfected with only GFP-Cb or Stx1 were mixed and then immunoprecipitated (2+3) with the antibodies indicated. (B) HeLa cells were transfected with syntaxin 1 (1) or co-transfected with syntaxin 1 and nSec1 (2). 24 hours after transfection, cells were lysed and processed for immunoprecipitation with either anti-syntaxin 1 mouse monoclonal antibody (1 and 2) or with control mouse immunoglobulins (1C and 2C). Proteins were resolved by SDS-PAGE, and western blots were probed with the antibodies indicated. Note that cellubrevin, SNAP-23 and TI-VAMP but not VAMP4, endobrevin or Vti1b were recovered in the syntaxin 1 immunoprecipitate. SM, starting material.