RhoB is up-regulated in response to TNF in primary human endothelial cells. (A) HUVECs were starved for 12 h and stimulated at different times with 10 ng/ml TNF. Right graphs quantify the expression changes of RhoA, RhoB, and RhoC in response to TNF. Mean + SEM of three different experiments. **, P = 0.001; *, P = 0.02 (B) HUVECs were left unstimulated or stimulated with TNF for 7 h, then fixed and stained with an antibody to RhoB and with phalloidin-TRITC to detect F-actin. TNF increased RhoB staining (enlarged areas). (C) HDMECs were stimulated with TNF at the indicated times and stained for the indicated proteins. (D) HUVECs were stimulated with TNF at different times, and RhoA, RhoB, and RhoC transcripts were analyzed by qPCR. Results were normalized to mRNA levels of β-actin and GADPH. Mean + SEM from four different experiments. Statistical significance tested for the three graphs *, P = 0.022. (E and F) Effect of 7-h TNF stimulation on active Rho proteins detected by pull-down assays. (F) Mean + SEM of six different experiments. No statistical significance with the Student’s t test was found for any data comparison in the three graphs. Bars, 20 µm.