Subcellular localization of endogenous Actα, Actβ, and Actγ mRNAs in cultured motoneurons. (A) In situ hybridization signals for Actα (A), Actβ (B), and Actγ transcripts (C) were detected in soma, axons, and axonal growth cones of cultured motoneurons. Cells were immunostained against Tau to determine cell boundaries. (D–F) Quantification of FISH signals shows that Actβ and Actγ are detectable at similar levels in the soma, but Actβ is more abundant in the axon and axonal growth cone (***, P < 0.0001 by one-way ANOVA with Dunn’s post-test for n = 6). Shown are mean ± SEM. (G) In triplex assays, cells were incubated simultaneously with probes detecting transcripts of Actα (white), Actβ (green), and Actγ (blue). Differential localization of actin mRNAs in soma, axons, and growth cones indicates that these mRNAs are present in different RNP granules. White circles and arrowheads in A and G indicate Actα mRNA punctae. Bars, 10 µm.