ASB2-induced FLNa degradation is dependent on ASB2 ubiquitin ligase activity and the proteasome. (A) Schematic representation of ASB2, ASB2LA, a BC box mutant, and ASB2-Δ, a deletion mutant lacking the SOCS box. The BC box mutation is indicated in bold. (B) ASB2-Δ and ASB2LA do not activate formation of polyubiquitin chains by the E2 enzyme. Elongin B, elongin C, cullin 5, and Rbx2 together with ASB2wt, ASB2LA, or ASB2-Δ were expressed in Sf21 cells. ASB2 complexes were immunoprecipitated (IP) with anti-flag or -ASB2 antibodies as indicated, and incubated with Uba1, Ubc5a, ubiquitin, and adenosine triphosphate. Their ability to stimulate polyubiquitination was assessed by Western blotting with antibodies to polyubiquitinylated proteins. ASB2 immunoprecipitation was assessed by blotting with anti-ASB2 antibodies. (C) ASB2 ubiquitin ligase activity is required for FLNa degradation. HeLa cells were transfected with GFP, GFP-ASB2wt, GFP-ASB2LA, or GFP-ASB2-Δ expression vectors and analyzed 24 hours after transfection using an antibody directed against the N-ter of FLNa. Scale bar represents 20 μm. (D) ASB2-induced FLNa degradation is dependent on proteasome activity. Ten hours after transfection with GFP-ASB2wt, HeLa cells were left untreated (−) or treated with 1 μM MG132 for 14 hours. Percentages of FLNa-negative GFP-ASB2-positive cells were counted 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) FLNa-GFP degradation by ASB2 is dependent on proteasome activity. NIH3T3 cells were transfected with FLNa-GFP, Myc-ubiquitin, and DsRed (−), DsRed-ASB2wt (wt), or DsRed-ASB2LA (LA) expression vectors. Seven hours after transfection, cells were left untreated or incubated with 1 μM MG132 or 20 μM LLnL for 17 hours, as indicated. A total of 20-μg aliquots of whole cell extracts were immunoblotted with antibodies directed against the N-ter of human FLNa and ASB2. Antibodies to human FLNa do not recognize endogenous mouse FLNa.