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. 2021 Mar 30;220(6):e202002084. doi: 10.1083/jcb.202002084

Figure S5.

Figure S5.

DQ-BSA labels degradative compartments in the endolysosomal pathway in hippocampal neurons. (A) Quantitation of the density of DQ-Green-BSA–positive puncta in dendrites and axons (mean ± SEM; dendrites, n = 34 neurons; axons, n = 49 neurons from three independent experiments; 13–14 DIV; unpaired t test; ****, P ≤ 0.0001). (B) Kymograph analysis of DQ-Green-BSA and DQ-Red-BSA in dendrites. Yellow arrowheads denote puncta positive for both DQ-Green-BSA and DQ-Red-BSA. Horizontal bar, 5 µm. Vertical bar, 10 s. (B’ and B”) Corresponding quantitation of the percentage colocalization (Coloc) between DQ-Green-BSA and DQ-Red-BSA in dendrites (mean ± SEM; n = 16 neurons from three independent experiments; 13–15 DIV). (C) Kymograph analysis of DQ-Red-BSA and early endosomes labeled with GFP-Rab5 in dendrites. Red arrowheads denote puncta positive for only DQ-Red-BSA, and green arrowheads denote puncta positive for only GFP-Rab5. Horizontal bar, 5 µm. Vertical bar, 10 s. (C’ and C”) Corresponding quantitation of the percentage colocalization between DQ-Red-BSA and GFP-Rab5 in dendrites (mean ± SEM; n = 11 neurons from two independent experiments; 13–15 DIV). (D) Kymograph analysis of DQ-Red-BSA and endocytic cargo BSA-488 in dendrites. Yellow arrowheads denote puncta positive for both DQ-Red-BSA and BSA-488, and green arrowheads denote puncta positive for only BSA-488. Horizontal bar, 5 µm. Vertical bar, 10 s. (D’ and D”) Corresponding quantitation of the percentage colocalization between DQ-Red-BSA and BSA-488 in dendrites (mean ± SEM; n = 8 neurons from two independent experiments; 14 DIV). (E) Maximum projections of Z-stacks of rat hippocampal neurons treated with 100 µM E64d and 30 µM pepstatin A versus DMSO for 3 h. During the last hour of treatment, neurons were also incubated with BSA-488 or DQ-Green-BSA. Inhibition of protease activity reduces DQ-Green-BSA fluorescence but has minimal effect on BSA-488. Scale bar, 20 µm. (F) Kymograph analysis of BSA-647 and DQ-Green-BSA in the axon. Red arrowheads denote puncta positive for only BSA-647, and green arrowheads denote puncta positive for only DQ-Green-BSA. Horizontal bar, 5 µm. Vertical bar, 10 s.