Fig. 3.
ANXA1 regulates tightness through an interaction with the actin cytoskeleton. (A) Rhodamine-phalloidin staining of F-actin in WT, FL11, and AS4 hCMEC/D3 clones reveals a disorganized cytoskeletal structure in AS4 cells. (Scale bar: 10 µm.) (B) Coimmunoprecipitation analysis of ANXA1 and β-actin interaction: Upper: Total cell extracts (250 µg) were immunoprecipitated with 5 μg of a monoclonal anti–β-actin antibody, and Western blot was performed with anti-ANXA1 antibody. Lower: Cellular extracts were immunoprecipitated with 5 µg of an anti-ANXA1 antibody, and Western blot was performed with an anti–β-actin antibody. (C) Confocal microscopic analysis of ANXA1 and β-actin colocalization determined by use of the proximity ligation assay (PLA; red), and F-actin identified by rhodamine-phalloidin staining (green) in WT hCMEC/D3 cells. Images are from a 400-nm optical section. (Scale bar: 5 µm.) (D) Confocal microscopic analysis of the actin cytoskeleton in isolated brain capillaries from WT and AnxA1−/− mice, identified by staining nuclei with DAPI (blue) and F-actin with rhodamine-phalloidin (red; typical images from n = 3 independent preparations). (Scale bar: 10 µm.)