Fig. 5.

N-terminal domain of drebrin localizes to the actin cytoskeleton and coprecipitates with cortactin. (A) DBN1 KO HEK293 cells stably expressing indicated GFP-tagged DBN1 constructs or control EGFP were analyzed by confocal microscopy for the localization of DBN1 (green), actin (red), and nucleus (DAPI, blue). Colocalization (yellow) is highlighted by white arrowheads. (Full-length: DBN isoforms A and E; N terminus: amino acid 1–366; middle region: 233–317; C terminus: 319–707). (Scale bar in panels and single z slices, 40 μm.) (B) DBN1 KO HEK293 cells stably expressing indicated GFP-tagged DBN1 constructs or control EGFP were subject to IP using α-GFP antibody and analyzed by Western blot using indicated antibodies. The IP band intensities were normalized to endogenous CTTN levels in IP input and compared with that of DBN-A (lane 1), which was set as 1.00. Bottom panels are 10% input. (C) Reconstituted DBN1 KO HEK293 cells were infected with RRV at MOI = 1 for 24 h and examined by RT-qPCR for viral NSP5 expression, normalized to that of GAPDH. For all figures, experiments were repeated at least three times. Data are represented as mean ± SEM. Statistical significance is determined by Student’s t test (***P ≤ 0.001).