Fig. 4

Establishment of NIH3T3-Dock180-ELMO1 cells. a NIH3T3 cells stably expressing extrinsic Dock180 were infected with retroviruses encoding wild-type ELMO1 and its Y724F mutant, and the introduced cells were selected with Blasticidin. Expression levels of extrinsic Dock180 and ELMO1 were examined by immunoblotting using Abs to Flag and ELMO1, respectively. b ELMO1 phosphorylation and Rac1 activation in NIH3T3-Dock180-ELMO1 cells. Trypsinized NIH3T3-Dock180-ELMO1 cells were replaced onto fibronectin-coated dishes and incubated for 30 min. To examine phosphorylation of ELMO1, immunoprecipitation and the subsequent immunoblotting were performed using Abs to ELMO1 and PY, respectively (upper two panels). For Rac1 activity, the cell lysates were subjected to the pull-down assay, followed by the immunoblotting using anti-Rac1 Ab (bottom two panels)