Figure 4.

Palladin is translationally regulated by mTOR signaling and localized in axons. A, Lysates of cortical neurons treated with control vehicle (DMSO) or Torin 1 (100 nm) for 24 h were immunoblotted with the palladin, phospho-S6, and S6 antibodies. The palladin intensity was quantified and normalized by the loading control S6. The average relative abundance of three replicates was indicated. B, Left, Lysates of cortical neurons transfected with control plasmid, palladin RNAi1, or palladin RNAi2 were immunoblotted with the palladin and ERK antibodies. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. Right, Lysates of HEK293T cells transfected with Flag-palladin together with control plasmid, palladin RNAi1, or palladin RNAi2 were immunoblotted with the Flag and ERK antibodies. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. C, Lysates of cortical neurons treated with control vehicle (DMSO) or Torin 1 (100 nm) for 24 h were immunoblotted with the Zfp36, phospho-S6, S6, and ERK antibodies. The Zfp36 intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. D, Lysates of cortical neurons treated with control vehicle (DMSO), Torin 1 (100 nm), Torin 2 (100 nm), or PP242 (2.5 μm) for 24 h were immunoblotted with the palladin, phospho-S6, and ERK antibodies. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. E, Lysates of cortical neurons treated with control vehicle (DMSO), Torin 1 (100 nm), or cytochalasin D (1 μm) for 2 h followed by 30 min treatment with puromycin were immunoblotted with the puromycin, phospho-S6, S6, and ERK antibodies. Relative abundance of nascent proteins labeled with puromycin was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. F, Lysates of cortical neurons treated with control vehicle (DMSO), Torin 1 (100 nm), or cytochalasin D (1 μm) for 24 h were immunoblotted with the palladin and ERK antibodies. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. G, Lysates of TSC1 mutant Neuro2A cells or control cells were immunoblotted with palladin, TSC1, phospho-S6, S6, and ERK antibodies. The schematic of mouse TSC1 gene indicates sites of mutations introduced with the CRISPR–Cas9 system. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. H, Lysates of cortical neurons from different time points were immunoblotted with the palladin and ERK antibodies. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated. I, Hippocampal neurons cultured for 5 d in vitro were subjected to immunocytochemistry with the palladin and βIII Tubulin antibodies. Scale bar, 5 μm. J, Sections of E16 mouse cerebral cortex were subjected to immunohistochemistry with the palladin antibody (green), Tau-1 antibody (red) and stained with Hoechst (blue). Regions in the boxes 1–4 are enlarged in the bottom panels. The palladin immunoreactivity is higher in the intermediate zone where axons reside compared with other parts of the cerebral cortex. Scale bar, 100 μm. K, Lysates of whole cells or isolated neurites of cortical neurons treated with control vehicle (DMSO) or Torin 1 (100 nm) for 12 h were immunoblotted with the palladin, phospho-S6, S6, ERK, and nucleoporin antibodies. The palladin intensity was quantified and normalized by the loading control ERK. The average relative abundance of three replicates was indicated in the graphs. Asterisks indicate p < 0.05 in the graphs.