Fig. 2. Cellular localization of Syt I and Syt II. (A) RBL cells transiently transfected with T7-Syt I, HA-Syt II or stably transfected with Syt II were grown on glass coverslips for 24 hours. Cells were subsequently labeled with anti-T7 monoclonal antibodies (a), anti-HA monoclonal antibodies (b) or anti-C2A-Syt IX polyclonal antibodies (c) followed by Cy3-conjugated donkey anti-mouse or Rhodamine-conjugated donkey anti-rabbit IgG. Cells were processed for immunofluorescent staining and visualized by confocal microscopy. (B) Subcellular fractionation of T7-Syt I or HA-Syt II-transfected RBL cells. Cell homogenates derived from RBL cells transfected with either T7-Syt I or HA-Syt II cDNA were fractionated on continuous sucrose gradients as described previously (Baram et al., 1999). Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-T7, anti-HA or anti-Gi₂ antibodies. Fractions were also examined for ß-hexosaminidase activity (presented as OD at 405 nm). Bar, 10 µm.