Dynein effectors associate with autophagosomes in the axon. (A and B) Micrograph and line scan demonstrating LC3 can colocalize with all three candidates simultaneously. Yellow dashed line indicates line scan. Scale bar, 1 µm. (C) Schematic illustrating PLA method. Bottom right; Representative negative control (no primary antibody). (D and E) Representative micrographs and quantifications of PLA puncta for endogenous LC3 with Halo-tagged JIP1, HAP1, and JIP3 along the axon (dotted gray line). Arrowheads indicate PLA puncta. Scale bar, 10 µm. Dashed gray line indicates negative control (missing primary antibody). n = 27–28 neurons. (F) Immunoblotting of autophagosomal isolation. (G) Quantification of lipidated LC3-II isoform compared with cytosolic LC3-I. n = 3 preps; two-tailed paired t test (P = 0.0254). (H) Quantification of enrichment in the autophagosomal fraction, displayed as relative to brain lysate (input). n = 3 preps; one-way ANOVA for each; Golgi protein GM130 (P = 0.2128), Htt (P = 0.0198). (I–L) Representative micrographs and quantifications of PLA puncta between endogenous DIC and Halo-tagged HAP1 (I and J) or JIP3 (K and L) along the axon (dotted gray line). Arrowheads indicate PLA puncta. Scale bar, 10 µm; n = 9 neurons/region; dashed gray line indicates negative control (missing primary antibody); one-way ANOVA. Bars throughout show mean ± SEM. *, P < 0.05.