Fig. 5
From: RNA localization is a key determinant of neurite-enriched proteome
Validation of local translation by imaging. a Scheme illustrating the principle of puro-PLA assay to visualize specific newly synthesize proteins34. b Puro-PLA images of selected newly synthesized proteins in iNeurons. LMNB1 was used as reference for somatically produced proteins34. For negative control, cells were pretreated with the protein synthesis inhibitor anisomycin (anisomycin), protein-specific antibody was omitted (α-puro only) or mock rabbit IgG was used instead of specific antibody (mock IgG-puro-PLA). Immunostaining with MAP2 (magenta) and NF (green) enables detection of dendrites (MAP2-positive neurites) and axons (NF-positive, but MAP2-negative neurites). COL3A1, MYO1C, CALD167 (Caldesmon), VCL68 (Vinculin), TAGLN69 (Transgelin), and PPFIBP1 are examples of neurite-translated proteins (Ribo-seq log2 neurites/soma = 2.4, 0.9, 1.2, 1.4, 3.7, and 2.1 correspondingly). Btz/CASC3 is a protein that showed no preferential translation in neurites (Ribo-seq log2 neurites/soma = −0.2). Magnifications of neurite sections (inserts) shown next to the images. Scale bar = 5 μm. Puro-PLA signal (white), NF (green), MAP2 (magenta), DAPI (blue). See also Supplementary Fig. 7 for puro-PLA validation on hippocampal neurons and different length of puromycin treatment
