Fig 2: An overview of organelles involved in the trafficking of newly-emerged TNR epitopes.a Newly-emerged TNR epitopes were labeled 12 h post-blocking, concurrently with the application of LysoTracker™ Green, to label acidic organelles. After a 6-h incubation, allowing for internalization, surface TNR was stripped with proteinase K. Neurons were imaged live (epifluorescence). Scale bar = 4 µm. >70% of internalized TNR is present in acidic organelles. N = 3 independent experiments, =4 neurons per datapoint. Data represent mean ± SEM, dot indicate individual experiments. b–g To identify the compartments containing internalized TNR, newly-emerged TNR epitopes were labeled 12 h post-blocking and allowed to internalize for 6 h, after which remaining surface-bound TNR was stripped with proteinase K. The neurons were fixed and immunostained with organelle markers. Shown are 2-color-STED images of TNR (magenta) and organelle markers (green): caveolin1, Rab11a (recycling endosomes), LAMP1 (lysosomes), TGN38 (trans-Golgi network) and calreticulin (ER). The right side of each panel shows zoomed views of the dashed boxes. Arrowheads indicate colocalizing signals. Scale bar = 2 µm (full images), 500 nm (zoomed images). g Quantification of % TNR spots colocalizing with organelle markers, compared to a negative control (using non-specific primary antibodies). TNR colocalizes significantly with ER, TGN, LAMP1, Rab11a and caveolin. N = 3 independent experiments, =10 neurons per datapoint. One-way ANOVA (F8,18 = 4.284, **p = 0.005), followed by Fisher’s LSD to compare all markers with ‘neg ctrl’ (Caveolin1: **p = 0.002; Rab5: p = 0.099; Rab7: p = 0.126; Rab11a: *p = 0.017; Rab11b: p = 0.169; LAMP1: **p = 0.005; TGN38: ***p < 0.001; Calreticulin: ***p < 0.001). Data represent mean ± SEM, dots indicate individual experiments. h A fraction of newly-emerged TNR localizes to dendritic Golgi outposts following endocytosis. Newly-emerged TNR epitopes (magenta) were labeled 4 h post-blocking, and allowed to internalize over 12 h. The neurons were fixed and immunostained with TGN38 to identify dendritic Golgi outposts78,79. Representative images, taken with confocal microscopy, are shown. Arrowheads indicate colocalizing signals. Scale bar = 2 µm. N = 4 independent experiments. Source data are provided in Source Data file.

Fig 3: Subcellular localization of the a1 and a3 subunits. A–F, immunofluorescence microscopy of fixed and permeabilized PC-3 cells. Left column, cells stained with antibodies to a1 (A–C) or a3 (D–F). Center column, staining with antibodies to LAMP-1 (A and D), TfR (B and E), or Ac45 (C and F). Right column, merged images of those in left and center columns, with coincident pixels colored yellow. Nuclei are stained with Hoechst 33258. Scale bars, 10 µm (A and B) or 20 µm (C–F). G, quantitation of co-incident staining. Images such as those in A–F were analyzed using the Coincidence function of Imaris software. Values are expressed as the percentage of pixels showing staining for a1 (open histograms) or a3 (gray histograms) that also stain for the subcellular marker indicated. Error bars represent standard deviation. The a3 isoform showed significantly greater (p < 0.01) co-localization with the late endosomal marker LAMP-1 (38 cells analyzed) than did a1 (26 cells analyzed), a1 co-localizing significantly more than a3 (p < 0.05) with the plasma membrane/endosome marker TfR (23 and 27 cells, respectively). Both a1 and a3 showed strong co-localization with subunit A and Ac45 (11–24 cells analyzed). *, p < 0.01; ***, p < 0.001. H, both a1 and a3 associate with Ac45. The a1 and a3 subunit isoforms were immunoprecipitated (IP) from detergent-solubilized PC-3 cell lysate, and co-precipitating proteins were analyzed by immunoblotting (IB). Precipitation with normal rabbit serum was used as a control for nonspecific binding. A sample containing the equivalent amount of cell lysate to that used in the immunoprecipitations was also run (Input). In the immunoblot of the A subunit, the asterisk indicates recognition of the heavy chain of the precipitating IgG. Ab, antibody.

Fig 4: Effect of sortilin depletion on the expression and distribution of PSAP in RCE-1 cells. (A) Representative immunoblot of PSAP in RCE-1 M (mock-depleted), RCE-1 and RCE-1 KD cells and the corresponding quantification of bands. Bars represent the means of relative optical density (R.O.D.) ± SEM from three independent experiments. (**) significant difference from control and RCE-1 (p < 0.05). Ponceau S staining were used as loading control. The full-lenght immunoblot is presented in Supplementary Fig. 3. (B) Representative immunofluorescence staining of PSAP and LAMP-1 in RCE-1 and RCE-1 KD cells. Quantification of co-localization of PSAP and LAMP-1 (MCC-M1); LAMP-1 and PSAP (MCC-M2). Values are expressed as the means of Manders colocalization coefficients 1 and 2 (MCC-M1 and MCC-M2, respectively) ± SEM. (**) significant difference from RCE-1 (p < 0.05). Scale bars = 10 µm.

Fig 5: Effect of sortilin depletion on the distribution and processing of Cathepsin D in RCE-1 cells. (A) Immunofluorescence staining of sortilin and CatD in the RCE1 cells. Confocal representative images and quantification of co-localization of sortilin and CatD (MCC-M1); CatD and sortilin (MCC-M2). The shown cells were taken from the framed area of the lower magnification image (right merge). (B) Representative immunofluorescence staining of CatD and LAMP-1 in RCE1 and RCE-1 KD cells. Quantification of co-localization of LAMP-1 and CatD (MCC-M1); CatD and LAMP-1 (MCC-M2). Values are expressed as the means of Manders colocalization coefficients 1 and 2 (MCC-M1 and MCC-M2, respectively) ± SEM. (*) significant difference from RCE-1 (p < 0.01). Scale bars = 10 µm. (C) Representative immunoblot of CatD in RCE-1 M (mock-depleted), RCE-1 and RCE-1 KD cells. Bands intensities of pro-CatD (52 kDa) and CatD (48 kDa) were quantified separately. Bars represent the means of the total CatD (pro-CatD + CatD), as relative optical densities (R.O.D.) ± SEM (upper histogram) and the means of pro-CatD percentage in each sample ± SEM (lower histogram) from four independent experiments. (*) and (**) significant differences (p < 0.01 and p < 0.05, respectively). Ponceau S staining were used as loading control. The full-lenght immunoblot is presented in Supplementary Fig. 2.