ASB2, but not ASB2LA, induces polyubiquitylation of FLNa. Recombinant ASB2/elongin BC/cullin5/Rbx2 complexes were purified as in Figure 2B. All samples contained purified Uba1, UbcH5a, and ubiquitin. The 3 components of the NEDD8 pathway were added as indicated. Purified FLNa was also provided as indicated and subjected to ubiquitylation. Aliquots of the reaction mixture were analyzed by Western blotting using anti-FLNa (upper panel) and anti-ASB2 antibodies (lower panel).