Localization of actin nodes and DAAM1 patches. (A) In untreated cells, DAAM1 antibody and phalloidin staining showed patchy distribution of DAAM1, which colocalized along the actin filamentous network. Boxed regions are enlarged on the right. (B) In live cells, and after 20 min of 800 nM LatA treatment, EGFP-DAAM1 localized with actin nodes. B1 and B2, enlarged views of the boxed regions. (C) In cells pretreated with 20 µM of SMIFH2 for 4 h then 800 nM LatA for 20 min, DAAM1 patches either did not form (C1) or did not colocalize with actin nodes (C2). (D) Colocalization analysis showed that the SMIFH2-LatA–treated samples show significantly less colocalization of EGFP-DAAM1 to actin compared with samples that only received LatA treatment. (N, number of cells for each sample). Error bars indicate SD. (E) Localization of DAAM1 on F-actin by superresolution microscopy. Left: F-actin staining of the entire cell, visualized by Alexa Fluor 633–phalloidin staining. The boxed region is enlarged: PALM image of photoactivatable PA-TagRFP-DAAM1 (middle) and overlaid with the F-actin network (right). Bars: (A–C) 5 µm; (B1, B2, C1, and C2) 2 µm; (E, left) 10 µm; (middle and right) 2 µm.