Knockdown of both ezrin and moesin impairs microcluster formation and antigen aggregation. (A) Hel-IgM A20 B cells stably expressing control or ezrin shRNA (left), transiently transfected with control or moesin siRNA (middle), or stably expressing control or ezrin shRNA and then transiently transfected with control or moesin siRNA (right) were lysed and analyzed by SDS-PAGE followed by immunoblotting with antiezrin, antimoesin, and antitubulin as indicated. (B–D) Hel-IgM A20 B cells expressing control shRNA, ezrin shRNA, moesin siRNA, or ezrin shRNA and moesin siRNA were settled onto lipid bilayers containing Hel as antigen (Ag). (B) Fluorescently labeled antigen was visualized by confocal microscopy after 10 min of interaction. (C and D) Contact area (C; **, P = 0.0047) and total antigen intensity within the contact area (D; **, P = 0.0018) were quantified after 10 min of interaction in individual cells. (E) Representative images of antigen microclusters in A20 B cells expressing control shRNA and control siRNA, ezrin shRNA, moesin siRNA, or ezrin shRNA and moesin siRNA on planar lipid bilayers. (F) Quantification of the number of antigen microclusters in cells expressing control shRNA and control siRNA (40 ± 3), ezrin shRNA (42 ± 3), moesin siRNA (38 ± 3), and ezrin shRNA and moesin siRNA (27 ± 3). ***, P = 0.0007. (G) Cells expressing control shRNA and control siRNA or ezrin shRNA and moesin siRNA were settled onto anti-IgM–coated plates for the indicated time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti–phospho-p44 and -p42 MAPK (Erk1 and Erk2) or antitubulin. Data are representative of at least two independent experiments. (C, D, and F) Error bars indicate mean ± SEM. Bars, 2 µm.