Figure 3
LPS induces VCAM-1 expression via c-Src. (A) Cells were pretreated with PP1, and then incubated with LPS for 8 h. VCAM-1 protein levels were determined. (B) Cells were pretreated with PP1, and then incubated with LPS for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. (C) Cells were transfected with c-Src siRNA, and then incubated with LPS for 8 h. The protein levels of c-Src and VCAM-1 were determined. (D) Cells were pretreated without or with PP1 (1 μM), and then treated with LPS for the indicated times or 10 min. The expression of phospho-c-Src was determined. (E) Cells were transfected with c-Src siRNA, and then stimulated with LPS for 30 min. The NADPH oxidase activity and ROS generation were determined. (F) Cells were transfected with c-Src siRNA, and then incubated with LPS for 30 min. The membrane fractions were prepared and subjected to Western blot using an anti-p47phox antibody. (G) Cells were treated with LPS for the indicated times. The cell lysates were subjected to immunoprecipitation using an anti-c-Src antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-TLR4, anti-p47phox, or anti-c-Src antibody. All figures are representative of three independent experiments, performed in duplicate. Data are expressed as means ± SEM. *P < 0.05; #P < 0.01, as compared with the cells exposed to LPS alone [A, B, D (lower panel)], scrambled siRNA alone (C, upper panel), LPS + scrambled siRNA [C (lower panel), E, F], or vehicle alone [D (upper panel), G].