Increased RhoA and Rac activity and dyregulated RhoA activation after stimulation with CXCL12 and CXCL13 in Eμ-TCL1^Tg;Rhoh^−/−CLL cells. (A) RhoA and (B) Rac activity determined by pull-down assays in WT versus Rhoh^−/−CLL samples (WT versus Rhoh^−/−CLL; mean ± SEM, *P < .05, Student t test, n = 3). Representative photomicrographs showing immunofluorescence staining and membrane localization of (A) RhoA (green), (B) Rac (green), and DAPI stained nuclei (blue) in fixed WT and Rhoh^−/−CLL cells. Representative dot plot analysis of RhoA and Rac distribution randomly selected from 3 to 4 microscopic fields at 600× magnification are exhibited and show increased membrane localization as indicated by arrowheads in Rhoh^−/−cells. Primary WT and Rhoh^−/−CLL cells were analyzed for activated and total RhoA protein after (C) CXCL12 and (D) CXCL13 stimulation. Top panels: activated (GTP-bound) RhoA; bottom panels: total RhoA protein. Bar graphs depict the relative ratio of GTP-bound to total protein obtained by densitometric measurement of the respective bands in blots of 2 independent experiments. These ratios were normalized to the unstimulated condition and indicate the fold increase after chemokine stimulation (gray bars: WTCLL vs black bars: Rhoh^−/−CLL; mean ± SEM).