ASB2-induced FLNb degradation is dependent on ASB2 ubiquitin ligase activity and the proteasome. (A) ASB2-induced FLNb degradation depends on ASB2 ubiquitin ligase activity. NIH3T3 cells were transfected with GFP-ASB2wt or GFP-ASB2LA expression vectors, replated on coverslips coated with 5 μg/mL fibronectin 5 hours after transfection, fixed 15 hours after replating, and analyzed using an antibody directed against FLNb. Scale bar represents 50 μm. (B) FLNb-GFP degradation by ASB2 is dependent on proteasome activity. NIH3T3 cells were transfected for 24 hours with FLNb-GFP together with the GFP (−), GFP-ASB2wt (wt), or GFP-ASB2LA (LA) expression vectors in the absence or presence of 1 μM MG132 for 18 hours, as indicated. (C) HEK293T cells were transfected for 24 hours with FLNb-GFP (left) or GFP-talin (right) together with the GFP (−), GFP-ASB2wt (wt), or GFP-ASB2LA (LA) expression vectors, as indicated. In panels B and C, 20-μg aliquots of whole cell extracts were immunoblotted with antibodies to GFP.