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Fig. 1. RBO facilitates FM1-43-dye endocytosis at the neuromuscular synapse. (A) Schematic of dye-loading, temperature and imaging protocols used at the larval NMJ. In protocol 1, FM1-43 was loaded for 2 minutes with 60 mM [K⁺] at 25°C and then unloaded for 2 minutes with 60 mM [K⁺] at 37°C. In protocol 2, following a 10-minute incubation at 37°C, dye was loaded for 2 minutes at 37°C and then unloaded for 2 minutes at 25°C. (B) Representative images of wild type (OR), syx^3-69 and rbo^ts single mutants, and the rbo^ts;syx^3-69 double mutant following protocol 1. (C) Top three panels show representative images comparing FM1-43 loading and unloading following protocol 2. Bottom panel shows rbo^ts;syx^3-69 37°C unloading after 5 minutes of loading at 25°C. Scale bars: 10 µm. (D) Quantification of loading (L) and unloading (UL) with both protocols. Error bars show mean ± s.e.m. ^***Significance of P<0.001.

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