FIGURE 5.

β-Arrestin 2 dynamically interacts with p38 after TLR4 triggering. A, WT and β-arrestin 2 KO MEFs were stimulated with LPS (20 μg/ml) for the indicated times. Levels of total and phospho-ASK1, total and phospho-MEK3/6 were determined by immunoblotting. B, peritoneal macrophages isolated from WT and β-arrestin 2 KO mice (n = 5 per group) were treated with 10 μg/ml LPS for the indicated times. The expression of total and phospho-p38, total and phospho-ASK1, total and phospho-MEK3/6 was examined as in A. C, WT MEFs and peritoneal macrophages were stimulated with LPS at the dose of 20 μg/ml and 10 μg/ml, respectively, for the indicated times. p38 (green) and β-arrestin 2 (red) distribution were viewed using confocal microscopy. Bar, 15 μm. D, control vector or β-arrestin 2 plasmid-transfected HEK293/TLR4 cells were cultured in the absence or presence of LPS (1 μg/ml) for the indicated times. Cell extracts were immunoprecipitated with anti-GFP antibody and then analyzed together with cell lysates by immunoblotting with indicated primary antibodies. IP and IB denote immunoprecipitation and immunoblotting, respectively. E, THP-1 cells, co-transfected with β-arrestin 2 plasmid and p38 plasmid, were cultured with LPS (1 μg/ml for 5 min), TNF-α (40 ng/ml for 5 min), or CpG-ODN (2 μm for 30 min). Interaction of β-arrestin 2 and p38 were determined as in (D). All of the results are representatives of three independent experiments.