MyD88 and TRAF6 are necessary for LPS-induced VCAM-1 expression. (A) RASFs were treated with various concentrations of LPS for the indicated time intervals and collected for Western blot analysis of VCAM-1 protein expression. (B) Cells were treated with LPS (100 μg·mL−1) for the indicated time intervals. Membrane fraction was prepared and examined by Western blotting. Gαs was used as a marker protein for the membrane fraction. (C) The levels of mRNA and VCAM-1 expression were analysed by real-time PCR and promoter luciferase activity respectively. (D) RASFs were transfected with TLR4, MyD88, TRAF2 or TRAF6 siRNA, and then treated with 100 μg·mL−1 LPS for (D) 6 h or (E) 4 h. (D) The protein expression of TLR4, MyD88, TRAF2, TRAF6 and VCAM-1 were determined by Western blot. (E) The levels of VCAM-1 mRNA and promoter activity were determined by real-time PCR and promoter activity. All analyses were performed on samples from three RA patients. Results are representative of three independent experiments. Values are the mean ± SEM. In B, #P ≤ 0.01 versus vehicle alone. In D, #P ≤ 0.01 versus LPS alone.