FIGURE 4:

Rac mediates ERK and RhoA activation induced by TNF-α. (A) LLC-PK₁ cells were transfected with NR siRNA or porcine Rac1/2 siRNA. Forty-eight hours later the cells were incubated in Na⁺ medium for 15 min, followed by the addition of 10 ng/ml TNF-α in Na⁺ medium or exchange of the medium for K⁺ medium (5 min). pERK was detected and quantified as in Figure 1. The blot was stripped and reprobed with anti-Rac. (B) Cells were transfected with HA-ERK2 with or without cotransfection of DN-Rac and 48 h later treated with TNF-α (5 min). HA-ERK was precipitated and its phosphorylation detected as in Figure 2D. (C) Cells were transfected with NR siRNA or porcine Rac1/2-specific siRNA for 48 h. Cells were treated with TNF-α (5 min), and active RhoA was precipitated with GST-RBD and quantified as described for Rac in Figure 3. The graphs show mean ± SE from n = 3 (A, B) or 5 (C) independent experiments.