Sephin1

Fluorescence recovery after photobleaching (FRAP) experiments were conducted at 37°C, 5% CO₂ as previously described (40 (link)). Briefly, dissociated adult mouse DRG cultures transfected with the GFP^MYR5’/3’khsrp were equilibrated in culture medium as above except phenol red was excluded. A region of interest (ROI) in the most distal axon of dissociated DRG neurons was photobleached with 488 nm argon laser set at 100% power for 80 frames at 0.65 s each. Pre-bleach and post-bleach signals were captured using 70% power for 488 nm laser line every 30 s (2 for pre-bleach and 30 for post-bleach). Translation dependence for recovery was tested by pre-treating DRG cultures with 100 μg/ml anisomycin (Sigma) or 150 μg/ml cycloheximide (Sigma) 20 min prior to photobleaching. For testing Ca²⁺-dependent translation by FRAP, transfected DRG cultures were pretreated with 1 μM thapsigargin (Sigma), 3 μM BAPTA-AM (Sigma), 90 μM GSK260614 (Bio-Techne Corp/Tocris, Minneapolis, MN) or 50 μM Sephin1 (Apexbio, Houston, TX). Leica SP8X confocal microscope was used for imaging at 37°C, 5% CO₂ with 63X/NA 1.4 oil immersion objective. Pinhole was set to 3 Airy units for pre-bleach, bleach, and post-bleach sequences to ensure full thickness excitation of the axon. ROIs were 40 × 40 μm and at least 250 μm from the soma.