ASB2 induces FLNa degradation. GFP-tagged ASB2 colocalizes with F-actin (A) and FLNa (B) in stress fibers (top), lamellae (middle), and filopodiae (bottom). HeLa cells were imaged 10 hours after transfection with GFP-ASB2 expression vector. Actin was detected with Alexa 568-phalloidin and FLNa with an antibody against its N terminus. Higher magnification views of the lower panels are also shown (bottom). (C) HeLa cells were transfected with GFP or GFP-ASB2 expression vectors as indicated; 24 hours later, FLNa was localized by staining the cells with antibodies directed against the N terminus (N-ter) or C terminus (C-ter) of the protein. Scale bars represent 20 μm. (D) FLNa-negative, GFP-ASB2-positive HeLa cells were counted 10 and 24 hours after transfection among a randomly selected pool of at least 100 cells expressing GFP-ASB2. Results are mean plus or minus SEM from 3 independent experiments. (E) NIH3T3 cells were mock-transfected or transfected with FLNa-GFP and GFP-ASB2 expression vectors for 24 hours, as indicated; 20-μg aliquots of whole cell extracts were immunoblotted with antibodies directed against the N-ter of human FLNa and GFP. Antibodies to human FLNa do not recognize endogenous mouse FLNa. (F) Triple staining showing F-actin organization and FLNa expression in HeLa cells expressing GFP or GFP-ASB2.