FLNa and FLNb are substrates of the ASB2α E3 ubiquitin ligase complex in DCs. (A) Expression of FLNa, FLNb, and α-actinin 1 was analyzed by western blot using 15-μg aliquots of whole-cell extracts of ASB2α^−/− and ASB2α^+/+ BMDCs from 2 independent cell cultures as indicated. (B) Expression of FLNa was assessed by immunofluorescence in ASB2α^−/− and ASB2α^+/+ BMDCs. Cells were harvested, centrifuged onto poly-l-lysine–coated glass coverslips, fixed, stained for FLNa and phalloidin, and imaged (left panel). Scale bar represents 20 μm. Dot plots show the overall distribution of relative FLNa fluorescence intensities, and lines show the median values (right panel). (C) Histograms show FLNa expression in ASB2α^−/− and ASB2α^+/+ BMDCs (left panel). The relative quantification of FLNa expression in ASB2α^−/− and ASB2α^+/+ BMDCs is shown as means and SD (middle panel). (Sample size: ^+/+ = 19; ^−/− = 26). Right panel shows the percentages of FLNa-negative and FLNa-positive BMDCs as means and SD. (Sample size: ^+/+ = 24; ^−/− = 26). (D) Relative expression of FLNa mRNAs in ASB2α^−/− and ASB2α^+/+ BMDCs assessed by quantitative real-time RT-PCR. Levels were normalized to Arbp. (E) Mouse ASB2α E3 ubiquitin ligase activity is required for FLNa degradation. NIH3T3 cells were transfected with GFP-mASB2α (ASB2α wt) or GFP-mASB2αLA (ASB2α mut) expression vectors and were analyzed 48 hours after transfection using an antibody directed against FLNa and phalloidin. Scale bar represents ***P < .001.