ASB2 induces degradation of FLNa and FLNb in differentiating myeloid leukemia cells. (A-C) Down-regulation of FLNa and FLNb in myeloid leukemia cells induced to differentiate correlates with ASB2 induction. NB4 and PLB985 cells were treated with 10⁻⁶ M of all-trans RA. Differentiation was assessed by cell morphology on May-Grünwald-Giemsa-stained cytospin slides (A). Expression of ASB2, FLNa, FLNb, and non-muscle myosin IIA (NM myosin IIA) was analyzed by Western blot using 20-μg aliquots of whole cell extracts (B) and by immunofluorescence (C). (D) ASB2 knockdown delayed RA-induced FLNa and FLNb degradation. Stable PLB985 cell populations expressing shRNAs directed against ASB2 (no. 1 and no. 2) or transfected with the empty vector (−) were treated with 10⁻⁶ M RA for different times as indicated; 10-μg aliquots of the protein extracts were analyzed by Western blotting. (E) PLB985 cell populations expressing shRNA no. 1 or transfected with the empty vector (−) were untreated and treated with RA. (Top panel) Representative experiment of 3 showing numbers of viable cells. (Bottom panel) Percentages of CD11b-positive cells. Error bars represent SDs from the results of 3 independent experiments.