Fig. 5. Effect of anti-Rab27B and anti-Slac2-c-SHD antibodies on the interaction between Rab27B and Slac2-c in vitro. (A) FLAG-Rab27B binding activity of T7-Slac2-c and T7-Slp4-a in the presence or absence of anti-Rab27B IgG. The FLAG-Rab27B beads (bottom panel) were incubated with T7-Slac2-c (lanes 1 and 2) or T7-Slp4-a (lanes 3 and 4) in the presence or absence of the anti-Rab27B IgG as described in the Materials and Methods, and the T7-tagged proteins trapped by the beads were analyzed by immunoblotting with HRP-conjugated anti-T7 tag antibody (1/10,000 dilution) as described previously (Fukuda et al., 1999). Note that the anti-Rab27B IgG specifically disrupted the Rab27B–Slac2-c interaction (lane 2, middle panel) but had no effect on the Rab27B–Slp4-a interaction (lane 4, middle panel). (B) Inhibition of the Rab27B–Slac2-c interaction by the anti-Slac2-c antibody. The T7-Slac2-c beads (bottom panel) were incubated with either the anti-Slac2-c IgG or a control rabbit IgG, and the FLAG-Rab27B trapped by the beads was analyzed by immunoblotting with HRP-conjugated anti-FLAG tag antibody as described previously (Fukuda et al., 1999; Fukuda et al., 2002b). Note that the anti-Slac2-c IgG, but not control IgG, inhibited the interaction between Rab27B and Slac2-c (compare lanes 2 and 3 in the middle panel). Input means 1/80 volume of the reaction mixtures used for the immunoprecipitation studies (top panels in A and B). The positions of the molecular mass markers (in kDa) are shown on the left.