Identification of the minimal region of ASB2α necessary for association with FLNa. A, HeLa cells were transfected with GFP, GFP-ASB2α(1–20), GFP-ASB2αANK(1–3), GFP-ASB2αANK(1–5), GFP-ASB2αANK(1–7), GFP-ASB2αANK (1–9), GFP-ASB2αANK(1–12), or GFP-ASB2ΔN/ANK(1–12) expression vectors and analyzed 8 h after transfection. B, HeLa cells were transfected with GFP-ASB2αANK(1–12) or GFP-ASB2αANK(1–10) expression vector. Eight hours after transfection, cells were pretreated with Triton X-100 before fixation and stained with anti-FLNa and phalloidin. C, ASB2αANK(1–10) associates with FLNa. Protein extracts from demembranated cells expressing ASB2αANK(1–10) were subjected to immunoprecipitation (IP) with anti-FLNa or control antibodies and then assessed for ASB2α or FLNa. Scale bar represents 20 μm.