Fig. 4.

TIPE2 inhibits the activation of Rac and its effectors. (A) Confocal microscopy of 293T cells expressing TIPE2-GFP or GFP alone. To detect active Rac, cells were stained with anti–GTP-Rac and Alexa Fluor 555-labeled goat anti-mouse Ig (red). Images are representative of three independent experiments. (Right) The percentages of GTP-Rac–positive cells in total GFP positive cells. A total of 70 cells were counted for each condition (B) 293T cells were transiently transfected with TIPE2 and/or HRas12V as indicated. Cell lysates were subjected to pull-down, using PAK-GST protein beads. Rac from the pull-down and total Rac in the lysates were detected by Western blot. (C) WT and Tipe2^−/− BMDMs were plated on uncoated or fibronectin-coated dishes for 15 min at 37 °C. Active and total Rac levels in the cells were determined as in B. (D) 293T cells were transfected with or without Myc-Rac161L (2 μg/6-cm dish) and increasing amounts of Flag-TIPE2 constructs (1–2 μg/6-cm dish) for 8 h, and the levels of phospho(p)-JNK, total JNK, phospho(p)-PAK, total PAK, Myc, and Flag were determined by Western blot. Bar graphs show relative p-JNK and p-PAK levels as determined by densitometry. (E) WT and Tipe2^−/− BMDMs were treated with LPS (100 ng/mL) for the indicated times and F-actin level was measured as described in Methods. The “% increase in F-actin” = [(F-actin in LPS-treated cells − F-actin in untreated cells)/F-actin in untreated cells] × 100. Results are means ± SEM. Statistics were performed on pooled data from three independent experiments. *P < 0.05, **P < 0.01.