FIG. 2.

Activated Rho and membrane-targeted Rho preferentially associate with PKCα. (A) Jurkat cells were transfected with 20 μg of pEBG (lanes 1, 3, 5, and 7) or V14Rho (lanes 2, 4, 6, and 8) for 15 h. Cells (5 × 10⁶) were lysed, precipitated with glutathione-Sepharose beads, and immunoblotted with anti-PKC antibodies (A) and anti-GST antibody (B). (C) Jurkat cells were transiently transfected with pEBG (lanes 1 and 2) or V14Rho (lanes 3 and 4) for 15 h. Cells (7 × 10⁶) were lysed in hypotonic solution and fractionated as described in Materials and Methods. Proteins that precipitated with glutathione-Sepharose beads were immunoblotted with anti-PKCα antibody (C) and anti-GST antibody (D). C, cytosol fraction; M, membrane particulate fraction. (E) Jurkat cells were transiently transfected with 20 μg of pEBG, wt Rho, V14Rho, N19Rho, or V14Rho-ΔCAAX for 15 h. Transfected cells (5 × 10⁶) were lysed, and glutathione bead-precipitated proteins were immunoblotted with anti-PKCα antibody (E) and anti-GST antibody (F).