FIG. 4.

Involvement of Rho family proteins in Ral signaling in PC12 cells. PC12 cells were transiently transfected with various expression constructs, and 24 h later the cells were treated with NGF. The cells were then processed as described in the legend to Fig. 1. (A) The cells were transfected with ΔN11Ral, and transfected cells were detected with α-Ral antibodies (left), or cells were transfected with Ral28N, and 3 days later the cells were treated with lysophosphatidic acid (2.4 μg/ml) for 90 min (right). (B) Cells were transfected with 61LCDC42 (left) or 61LRac1 (right). Three days later, the cells expressing GTPases were detected with either rabbit α-CDC42 or α-Rac1 antibody. (C) Cells were transfected with MYC-Rgr and activated 61LCDC42. Three days later, the cells were fixed and labeled with both rabbit α-CDC42 antibodies and mouse α-MYC monoclonal antibodies. CDC42 was detected with FITC-labeled anti-rabbit secondary antibodies (left), and Rgr was detected with TRITC-labeled anti-mouse secondary antibodies (right). (D) Cells were transfected with Rgr and 61LRac1 and processed as for panel B except that 61LRac-expressing cells were detected with rabbit α-Rac antibodies. (E) Cells were transfected with Ral28N plus MYC-17NCDC42. Three days later transfected cells were stained with both rabbit α-Ral antibodies and mouse anti-MYC antibodies. Ral28N expression was detected with FITC-labeled anti-rabbit secondary antibodies (left), and Rgr was detected with TRITC-labeled anti-mouse secondary antibodies (right). (F) Cells were transfected with Ral28N plus MYC-17NRac1 and processed as for panel E.