Fig 2: Analysis of endosomal Rab RNAi screen and the localization of Rabs to REs AIFM images of HeLa cells to study the localization of KIF13A‐YFP to REs.BPCR analysis of Rab‐knockdown HeLa cells to measure the knockdown efficiency of each shRNA as indicated. DNA band intensities were quantified and indicated on the gels.CSubcellular fractionation of HeLa cells to study the localization of different Rabs (red box) with respect to STX13 or AP‐1.D, EIFM analysis of HeLa cells that were transfected with different constructs as indicated.FLive cell imaging of mCherry‐Rab22A with respect to GFP‐Rab9A in HeLa cells. Arrowheads point to the Rab22A‐positive tubular structures. Magnified view of insets (at 0, 16, 40 s) are shown separately. Scale bars: 10 μm.GIFM images of Rab22A sh, BLOC‐1 sh and control sh HeLa cells that were transfected with indicated constructs.HGraphs represent the measurement of corrected total cell fluorescence (CTCF) in HeLa cells of Fig 2A. n = 6–8 cells. Average CTCF values (AU: arbitrary units) and their respective fold changes (mean ± SEM) are indicated.Data information: In (A), arrowheads and arrows point to the KIF13A‐positive tubular REs and E/SEs, respectively. In (D, E, G), arrowheads point to the STX13‐ or KIF13A‐positive tubular REs or E/SEs. Scale bars: 10 μm.

Fig 3: BACE1 is directed to late endosomes/lysosomes in Mgat3−/− cells BACE1 from 2-week-old mouse brains was immunoprecipitated and immunoblotted (left). Immunoprecipitated BACE1 activity was measured in vitro (right, n = 3). The values of Bace1−/− samples were subtracted as a background. N.S., P = 0.101. MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, APP, rab5, or rab7. EE, early endosome; LE, late endosome. Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, rab5, or rab9. EE, early endosome; LE, late endosome. Signal intensities were quantified and are shown in the right graphs. Immunostaining of 12-month-old mouse cerebral cortex for BACE1 and APP. A typical image in the vicinity of plaque-forming area is shown for hAPP/Mgat3+/+ brain. The area of co-localization was quantified using random images of cerebral cortex (n = 10). *P = 0.015. Immunoblot of BACE1 or GAPDH (loading control) from immortalized MEFs treated with a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine; CQ). Immunostaining of 12-month-old mouse cerebral cortex for BACE1 (F), or nicastrin (G), and Lamp1. LE, late endosome. Scale bar, 10 μm. The area in which co-localized staining was observed was quantified as a percentage of the total BACE1-positive area (right, n = 10). **P = 0.007. Data information: All graphs show means ± SEM (Student's t-test).Source data are available online for this figure.

Fig 4: Changes in retrograde transport kinetics of CDMPR to the TGN upon silencing Vps26, Rab9a, or TIP47.(A, B, C, D) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of Vps26 (A) or Rab9a (C) as described in Fig 1. The cells were labeled with [35S]sulfate for up to 75 min in the presence of 2 μg/ml VHH-2xTS. The nanobodies were isolated by Ni/NTA beads and subjected to SDS-gel electrophoresis followed by Western-blot (WB) analysis (anti-His6) and autoradiography ([35S]). In parallel, aliquots of the cell lysates were immunoblotted for actin as a control for the amount of cells used. Experiments as shown in panels (A) and (C) were quantified in panels (B) and (D), respectively, and presented as the percentage of the value of control-kd cells after 75 min (mean and SD of three independent experiments; two-sided t test: *P < 0.05; **P < 0.01). Control-kd is shown as black squares and target-kd as gray circles; uptake as open symbols, sulfation as filled symbols. (E, F) HeLa cells stably expressing His6/myc-tagged cathepsin D or PAUF were transfected with non-targeting control siRNA or RNAs silencing expression of Vps26 or Rab9a. Cells were incubated in serum-free medium supplemented with 5 mM mannose-6-phosphate for 2 h. Secreted procathepsin D (pCatD) or PAUF were collected by Ni/NTA beads and analyzed by immunoblotting with anti-myc antibodies. (G, H) Missorted procathepsin D or PAUF was quantified from immunoblots as shown in panels C and E, respectively, and normalized to the values of control knockdown cells (mean and SD of four [pCatD] and three [PAUF] independent experiments). (I, J) Cells stably expressing EGFP-CDMPR were transfected with non-targeting siRNA (control-kd) or with siRNA silencing expression of TIP47 and assayed and quantified as described above in panels (A, B, C, D) (mean and SD of three independent experiments).

Fig 5: Pancreatitis increases cytosolic Rab9 complex formation with RabGDI and stimulates Rab9 proteasomal degradation. (A–D) Pancreas cytosolic fractions from control animals and those subjected to CER-AP (4 hours) were resolved on a Superdex S200 gel-exclusion column using SMART system (details in Methods). Data are representative of 2 experiments on different animals, with similar results. (A) Eluted fractions were analyzed by IB using antibodies against Rab9 and RabGDIα/β. Samples were run on 3 gels (see full gels in Figure 3), and all the IBs were developed together. Dashed lines denote the first lane in each gel; narrow white space indicates omitted lanes in which protein size ladder was run (see Figure 3). (B and C) Band intensities of Rab9 and RabGDI in each fraction were densitometrically quantified and presented as % of maximal for each protein. (D) Amount of Rab9 in complex with RabGDIα was quantified as a ratio of sum of Rab9 band intensities co-fractionated with RabGDIα to that of total Rab9 (in all fractions 12–33) and presented relative to saline control. In the control (saline-treated) group, the bulk of Rab9 complex with RabGDIα eluted in fractions 22–24; in CER-AP, in fractions 20–25. (E and F) Acinar cells incubated for 30 minutes with and without the proteasomal inhibitor MG132 (50 μmol/L), followed by 30-minute incubation with and without 100 nmol/L CCK. Rab9 level was measured by IB. Values are mean ± SEM from 3 cell preparations. ∗∗∗P < .001 vs control. ###P < .001 vs CCK alone (no MG132). Significance was determined by 2-tailed Student t test (D) or 1-way ANOVA, followed by Tukey multiple comparisons test (F).