FIG. 6.

RhoH does not inhibit exchange factor TIAM-1 and binds to Rho GDP dissociation inhibitors. (a) A 2-μg sample of TIAM-1 was cotransfected with RhoH (1, 2, or 4 μg) by electroporation into Jurkat cells, and 24 h later, the cells were harvested and measured for levels of GTP-Rac with the PBD assay. An increased level of GTP-Rac was seen in TIAM-1-transfected cells. Cotransfection of RhoH did not change the level of GTP-bound Rac. (b) Myc-tagged Rho GDI-α, -β, -γ, and−1.6 (used as a control) were cotransfected into 293 cells with HA-tagged RhoH or HA-tagged Oct2 (used as a control). At 24 h after transfection, cell lysates were prepared from transfected cells and immunoprecipitation (IP) was carried out with anti-HA antibody or immunoglobulin G (used as a control). Immunoprecipitates were separated and transferred to a Western blot. The filter was then probed with anti-HA and anti-myc epitope antibodies. The top of panel b shows the specific immunoprecipitation of HA-RhoH (27 kDa) or HA-Oct2 (75 kDa) protein. Equal levels of protein reflect uniformity of transfection and immunoprecipitation. The lower part of panel b shows the coprecipitation of only Rho GDIs with HA-RhoH. Note the equal levels of Rho GDIs, indicating equal avidity of binding between RhoH and the three different Rho GDIs.