Characterization of the kinesin-1–dependent transport machinery in MCs. (A) Relative quantification of Rab27a, Rab27b, Slp1, Slp2, Slp3, Slp4, and Slp5 transcripts in BMMCs, using real-time PCRs. Transcript levels for each sample were expressed as a proportion of the mean value for Rab27b. (B) BMMCs were lysed and then analyzed by Western blotting with anti-Kif5b, anti-Slp2, anti-Slp3, and anti-Rab27 antibodies. (C) IgE-sensitized WT BMMCs were either not stimulated (NS) or were stimulated with 20 ng/ml DNP-HSA for 10 min and then lysed. Cell lysates were immunoprecipitated (IP) with a polyclonal anti-Rab27 antibody or with an isotype control antibody. The immunoblots were analyzed using anti-Kif5b, anti-Slp3, and anti-Rab27 antibodies. (D) Flag-Rab27b and GFP-Slp3 were coexpressed in HEK 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and then separated by SDS-PAGE. Coprecipitated Rab27b and Slp3 were immunoblotted with anti-Flag and anti-GFP. (E) Flag-Rab27b, -Slp3, and GFP-KLC1 were coexpressed in HEK 293T cells. Cells were either not stimulated or were stimulated with PMA/ionomycin, and then cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and separated by SDS-PAGE. Coprecipitated Rab27b or Slp3 and KLC1 were immunoblotted with anti-Flag and anti-GFP. (F) Flag-Rab27b, -Slp3, and GFP-Kif5b were coexpressed into HEK 293T cells. Cells were either not stimulated or were stimulated with PMA/ionomycin, and then cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and separated by SDS-PAGE. Coprecipitated Rab27b or Slp3 and Kif5b were immunoblotted with anti-Flag and anti-GFP antibodies. Data in C–F are representative of three independent experiments. (G) Schematic diagram of the heterotrimeric protein transport complex involved in the translocation of SGs upon activation.