Fig 2: BDNF-induced formation of ribosomal complexes can be visualized by proximity ligation assay. a Representative images show PLA signal from RPL24 and RPS6 proteins in growth cones of Smn+/+;SMN2tgtg and Smn-/-;SMN2tgtg motoneurons following 1-min BDNF stimulation. b Increased PLA signal is detectable in growth cones of BDNF-stimulated Smn+/+;SMN2tgtg (****P < 0.0001; n = 65–69 cells from 4 independent experiments), but not in Smn-/-;SMN2tgtg neurons (n.s., P = 0.9315; n = 39 cells from 3 independent experiments). As a negative control, Smn+/+;SMN2tgtg neurons were pretreated with puromycin before and during the BDNF pulse (a). Only a low PLA signal is detectable (n.s., P = 0.6882; n = 13 cells from 1 experiment). c Omitting either the RPL24 or the RPS6 primary antibody abolishes the PLA signal. Growth cones were outlined using GFP (see the method part for PLA assay). Data are presented in scatter dot plot; error bars represent mean ± SEM. Statistical analyses were done by Two-way ANOVA with Tukey multiple comparison post-hoc test

Fig 3: Detection of ribosomal subunit interaction by PLA and two-color dSTORM. a Representative images of RPL24/puromycin-PLA signal detected in growth cones of Smn+/+;SMN2tgtg motoneurons. b PLA signal for RPL24/puromycin increases after 1-min BDNF stimulation (***P = 0.0001; n = 53–57 cells from 3 independent experiments). In control experiments, anisomycin treatment (a) decreased the RPL24/puromycin-PLA signal (****P < 0.0001; n = 18 cells from 1 experiment). c No PLA signal is detectable in the absence of primary antibodies to RPL24 or puromycin. Growth cones were outlined using GFP (see the method part for PLA assay). d and e Representative two-color dSTORM images labeling RPL24 and RPS6 (d) and Y10B and eEF2 (e) in growth cones of Smn+/+;SMN2tgtg neurons. Upon 1-min BDNF pulse, RPL24 and RPS6 as well as Y10B and eEF2 form clusters that tightly colocalize within growth cones. Arrows indicate positions where the RPL24/RPS6 and eEF2/Y10B signals are located in proximity of 50 nm or less. Data in b are presented in scatter dot plot; error bars represent mean ± SEM. Statistical analyses were done by one-way ANOVA with Dunn’s post-hoc test