Actα protein localizes to axonal branch points, whereas Actβ and Actγ are highly enriched in axonal filopodia and axonal growth cones. (A–C) Immunoblots of total lysates obtained from actin isoform–specific knockdown and control motoneurons probed with specific Actα (A), Actβ (B), and Actγ (C) antibodies. Calnexin was used as loading control. All three isoforms are detected in cultured motoneurons. (D) shRNA-mediated knockdown leads to a 60% reduction in Actα (**, P < 0.004 for n = 4), a 95% reduction in Actβ (**, P < 0.002 for n = 10), and a 85% reduction in Actγ (**, P < 0.005 for n = 5) protein levels (one-tailed Mann-Whitney test). Shown are mean ± SEM. (E–G) Motoneurons were stained against Tau and Actα (E), Actβ (F), and Actγ (G). Actα protein is highly enriched in axonal branch points and neurites (E). Actβ (F) and Actγ proteins (G) are very abundant in the soma and localize to axonal filopodia and axonal growth cone filopodia. (H–J) Motoneurons were extracted using Triton X-100 to remove G-actin. Cells were fixed and stained with phalloidin and Actα (H), Actβ (I), and Actγ (J) antibodies. (H) Colocalization of Actα with phalloidin shows that this isoform incorporates into F-actin in the axon and particularly in the axonal branch points. (I) Filamentous Actβ is present predominantly in the axonal growth cone filopodia. (J) Actγ colocalizes with phalloidin mostly in the soma and axons. Images are representative of three independent experiments. Bars, 10 µm.