Figure 8A. Schematic representation of the experimental paradigm. Plasmid concentrations used: 1μg/μL pCS2-mRFP; 0.7μg/μL pCS2+Venus-no 3'UTR or pCS2+Venus-3'UTR of interest. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated. Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons. Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). , isolated axons (C) were used for FRAP analysis. Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). isolated transfected axons (D) were used for FRAP analysis. Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). In vivo (E), whole embryos with exposed brain were used. The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution. (E) The red area on the brain schematic indicate Sema3A expressing territories. Numbers of axons analyzed are reported between brackets. Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. APP, Amyloid beta precursor protein Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. THBS1, Thrombospondin 1 H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.