Figure 3. The cc2 domain of p115 binds Rab1b and is required to rescue Rab1b membrane association. (A) Schematic diagram of p115 domains. (B) Equivalent amounts (approximately 5 µg) of bead-bound GST (lane 7) and the indicated p115 constructs (see Materials and Methods) were incubated with 0.6 µg of purified hexahistidine-tagged Rab1b in the absence of GDP or GTPγS. Recovery of the Rab1b construct was determined by immunoblotting with an anti-Rab1b antibody and compared with the loading controls as indicated. (C-R) Cells were mock transfected or transfected with p115 siRNA and re-transfected after 48 h with plasmid encoding bovine p115Δcc1 (C-J) or Myc-tagged bovine p115Δcc2-C (K-R). After an additional 24 h, the cells were stained using rabbit anti-Rab1b antibody and either anti-bovine p115 antibody or anti-Myc antibody. Bar = 10µm. (S-T) The average signal intensity of Golgi membrane associated Rab1b for mock or p115 knockdown cells in the absence or presence of expressed p115Δcc1 (S) (mean ± SEM; > 20 cells) or p115Δcc2-C (T) is shown (mean ± SD; n = 2; > 20 cells for each experiment).