ICAM-1 Dynamics in Endothelium Figure 5. ICAM-1 requires filamin and its intracellular tail for the actin link and its motility. (A) TNF-a-pretreated HUVECs were cultured on FN-coated surfaces and filamin expression was reduced using siRNA, according to Kanters et al. [22]. Next, anti-ICAM-1-coated magnetic beads were allowed to adhere to the endothelium for 30 minutes, and samples were processed as described in the legend of figure 2C. Panels on the left show magnetic beads pull down. ICAM-1 was precipitated equally in both conditions. Clustering of ICAM-1 induced the link to actin, which was prevented by silencing Filamin A. Panels on the right show the expression of indicated proteins in the cell lysates. (B) Full length (FL) and DC ICAM-1- GFP are expressed in M2 and A7 cells. Panel shows FRAP quantification. The data show that the recovery of full length ICAM-1 is faster in the absence of filamin, e.g. in M2 cells. ICAM-1-DC-GFP FRAP is independent on the presence of filamin. Experiment is done three times. Data are mean 6 SEM. PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11336