CD81 is important for the formation of Rac-dependent actin membrane protrusions. Microscopic visualization of F-actin by the use of Cy3-phalloidin in BM-DCs after transfection with control siRNA (A-B) or CD81 siRNA (D-E). Corresponding differential interference contrast images to panels B and E, respectively (C,F). Quantification of membrane protrusions in LPS-stimulated Mo-DCs (G) and BM-DCs (I-J) on 2-dimensional fibronectin-coated surfaces, respectively. Quantification of membrane protrusions after overexpression with wild-type Rac1, constitutive active or dominant-negative Rac1 mutant (Q61L and T17N, respectively), all linked to EGFP (J). In each individual experiment, 200 mature phalloidin-stained BM-DCs were counted by the use of fluorescence microscopy (G, I-J). Measurement of F-actin by flow cytometry after Alexa 488-phalloidin staining of LPS-stimulated Mo-DCs (H). Analysis of the basal activities (GTP loading) of the small GTPases Rac or RhoA after RNAi of CD81 using commercial enzyme-linked assay (K-L). All shown fluorescence staining images (A-B,D-E) are confocal images, focused to the basal plasma membrane of the respective cells. Bars represent 5 μm. Each single experiment was performed in duplicate. Each experiment was repeated at least 3 times independently. Error bars indicate ± SD. *P ≤ .05, ***P ≤ .001.