Fig. 2. Localization of Rab27–Slac2-c and Rab27–Slp4-a complexes on secretory granules in rat parotid glands. (A) In vivo formation of Rab27–Slac2-c and Rab27–Slp4-a complexes in rat parotid glands. Immunoprecipitation with anti-Slp4-a, anti-Slac2-c, or anti-Syt I IgG was performed as described in the Materials and Methods. Co-immunoprecipitated Rab27A/B was detected by immunoblotting with anti-Rab27A/B specific antibody (12.5% non-reduced gels; solid arrowheads in the top and middle panels). Immunoprecipitated Slp4-a, Slac2-c and Syt I were separately visualized by immunoblotting with specific antibodies (10% reduced gels; open arrowheads in bottom panel). The asterisks in lane 3 indicate the degradation products of Slac2-c. Note that both anti-Slp4-a and anti-Slac2-c IgGs, but not Syt I IgG, immunoprecipitated Rab27A and Rab27B (lanes 2 and 3 in the top and middle panels). The positions of the molecular mass markers (in kDa) are shown on the left. (B) Localization of Rab27A, Rab27B, Slac2-c and Slp4-a proteins in secretory-granule-enriched fractions. Subcellular fractionation of the rat parotid glands was performed as described previously (Imai et al., 2003). A 25 µg amount of total homogenate (lane 1), apical plasma membrane (lane 2), and secretory granule membrane (lane 3) from rat parotid glands was loaded on 7.5% (for Slp4-a and Slac2-c) or 12.5% SDS-PAGE (for Rab27A/B) and immunoblotted with anti-Rab27A (top panel), anti-Rab27B (second panel), anti-Slp4-a (third panel), or anti-Slac2-c antibody (bottom panel). Note that both the Rab27A and Rab27B proteins were highly enriched on the secretory granule membranes, whereas the Slp4-a and Slac2-c proteins were localized on both the apical plasma membrane and the secretory granule membrane. The positions of the molecular mass markers (in kDa) are shown on the right.