The C-terminal domains of RhoH are required for its function. (A) Schematic representation of RhoH mutants. Numbers indicate amino acid position within the sequence. (B) Subcellular localization of RhoH deletion mutants. WT LDBM cells were transduced with retroviral vectors expressing EGFP-tagged WT or C-terminal–deleted RhoH constructs (EGFP-RhoH, EGFP-RhoHΔPR, EGFP-RhoHΔCT). Sorted EGFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (100 ng/mL) for 30 seconds, fixed, and stained with Alexa Fluor 550–labeled CTxB (red) and 4′,6-diamidino-2-phenylindole (DAPI). (C) Subcellular localization of RhoH deletion mutants in 32D cells. 32D cells were transduced with HA-tagged RhoH-YFP, RhoHÁPR-YFP, or RhoHÁCT-YFP. Sorted YFP⁺ 32D cells were fractionated into cytosolic fraction (S), detergent-soluble membrane fraction (P), detergent-insoluble cytoskeleton-enriched membrane fraction, and nuclear fraction. RhoH was detected with anti-HA antibody. (D) C-terminal prenylation site and polybasic domain of RhoH are required for its inhibition of cortical F-actin assembly. WT LDBM cells were transduced with retroviral vectors expressing RhoH-YFP, RhoHΔPR-YFP, or RhoHΔCT-YFP. Sorted YFP⁺/c-Kit⁺ cells were stimulated with SDF-1α (0 or 100 ng/mL) for 30 seconds before being fixed and stained with rhodamine-labeled phalloidin (red) and DAPI (blue). A total of 200 cells were counted under fluorescent microscope. Data represent the percentage of cells with cortical F-actin as the mean plus or minus SD; n = 3.