|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 6. Interaction of Dock180 with Crk is not required for Dock180-dependent Rac1 activation by RhoG. (A) The Dock180 constructs used in this study (Docker, Dock180 homology domain involved in exchange on Rac; Pro, proline-rich region; ISP, specific amino acid residues required for the activation of Rac within Docker domain; numbers indicate amino acid position within the sequence). (B) Flag-tagged Dock180 or Flag-tagged Dock180-T1657 expressed in HEK 293T cells was used in a pull-down assay with GST or GST-fused CrkII. Bound proteins and total cell lysates were analyzed by immunoblotting with anti-Flag antibody. (C) Lysates from HEK 293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Myc antibody, and bound proteins and total cell lysates were analyzed by immunoblotting with antibodies against Flag, HA and Myc. (D) Cellular homogenates from HEK 293T cells transfected with the indicated plasmids were separated into cytosolic (C) and membrane (M) fractions, and were then analyzed by immunoblotting. (E) Cell lysates from HEK 293T cells transfected with the indicated plasmids were incubated with GST-CRIB, and bound Rac1 was detected with anti-Rac1 antibody. Relative Rac1 activity was determined by the amount of GTP-bound Rac1 bound to GST-CRIB normalized to the amount of Rac1 in cell lysates analyzed by NIH Image software, and the value from untransfected cells was arbitrarily set at 1. Data are the means ± s.e.m. of three independent experiments. WT, wild-type.
|
