Fig. 4. The R30D mutation does not perturb other known coronin 1B molecular interactions. (A) HEK293 cells were co-transfected with Myc-tagged coronin 1B and various GFP-tagged coronin 1B mutants: wild type (WT), R30D mutant, coil-coiled domain (CC, 451-489) alone or a deletion mutant lacking the coil-coiled domain (CC, 1-450). We immunoprecipitated Myc-tagged coronin 1B with Myc antibodies and blotted with either anti-GFP or Myc antibodies. The results demonstrate that the coiled-coil domain of coronin 1B is both necessary and sufficient for oligomerization and that the R30D mutation does not affect oligomerization. (B) Rat2 fibroblasts were infected with the knockdown/rescue lentivirus expressing the coronin 1B shRNA and either wild-type coronin 1B or the R30D mutant. Cells were subjected to the in vivo phosphorylation and dephosphorylation assay described in Materials and Methods. (C) In vitro direct-binding assay using purified coronin 1B immobilized on Ni-NTA beads and purified bovine Arp2/3 complex in solution (5 nM). (D) Bound Arp2/3 complex on beads from experiments described in C was quantified by densitometry and normalized to the amount of coronin 1B. Results from three independent experiments are presented as the mean ± s.d. (E) Cells as described in B were lysed and GFP-tagged coronin 1B was immunoprecipitated using anti-GFP antibodies. To visualize the co-immunoprecipitated Arp2/3 complex, the R30D lane was intentionally overloaded. Arrowhead, GFP-tagged coronin 1B; arrow, endogenous coronin 1B.