Fig 2: TRIM39 decreases the ubiquitination of Rab7 at lysine 191 residue.a Vector or FLAG-TRIM39 was co-transfected with HA-ubiquitin into HCT116 cells for 48 h. Cell lysates were extracted and immunoprecipitated using an anti-Rab7 antibody. Then the precipitates were examined with anti-HA. b shNC and TRIM39 stably knocked down HCT116 cells were transfected with HA-ubiquitin for 48 h. Cell lysates were extracted and immunoprecipitated using an anti-Rab7 antibody. Then the precipitates were examined with anti-HA. c Myc-Rab7K191R was co-transfected with HA-ubiquitin into shNC or TRIM39 stably knocked down HCT116 cells for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA and anti-Myc antibody. d GST-RILPRBD fusion protein immobilized on glutathione-Sepharose beads were incubated with Myc-Rab7WT or Myc-Rab7K191R overexpressed HCT116 cell lysates at 4 °C for 4 h. Rab7 and GST were detected in the washed beads by western blot. e shNC and TRIM39 stably knocked down HCT116 cells were transfected with Myc-Rab7K191R for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-GDI2 and anti-Myc antibody. f HCT116 cells were transfected with Myc-Rab7WT or Myc-Rab7K191R for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-MON1A and anti-Myc antibody. g shNC or TRIM39 stably knocked down HCT116 cell lysates were extracted and immunoprecipitated with anti-Rab7 antibody. MON1A was detected by western blot.

Fig 3: Rab7 is a binding partner of TRIM39.a HEK293T cells were transfected with empty vector or FLAG-TRIM39 for 48 h. Total cell lysates were subjected to immunoprecipitation with anti-FLAG antibody. Rab7 was identified via mass spectrometry. b HEK293T cells were transfected with FLAG-TRIM39 for 48 h. Total cell lysates were immunoprecipitated with anti-FLAG or anti-Rab7 antibodies. Rab7 and FLAG-TRIM39 were detected by western blot. c, d GST-RILPRBD fusion protein immobilized on glutathione-Sepharose beads were incubated with Vector/FLAG-TRIM39-overexpressed LoVo cell lysates (c) or shNC/TRIM39 stably knocked down LoVo cell lysates (d) at 4 °C for 4 h. Rab7 and GST were detected in the washed beads by western blot. e, f Control (Vector) and FLAG-TRIM39-overexpressed LoVo cell lysates (e) or shNC and TRIM39 stably knocked down LoVo cell lysates (f) were extracted and immunoprecipitated using an anti-Rab7 antibody. Then the precipitates were examined with anti-GDI2. g shNC and TRIM39 stably knocked down LoVo cells were co-transfected with GFP-Rab7 and mCherry-LAMP1 for 48 h. Representative confocal microscopy images are shown. Scale bar, 5 µm. h Quantification of co-localization efficient and Pearson correlation of GFP-Rab7 and mCherry-LAMP1 treated as in g. 10 cells were analyzed. Data are shown as mean ± SEM. One-way ANOVA. **P < 0.01.

Fig 4: Knockdown of Rab7 significantly alleviated the symptom of CKD in vivo. (A, B) Sirius red staining was used to detect the fibrosis in kidney tissues of mice. The level of CD34 in kidney tissues of mice was detected by IHC staining. (C) The protein levels of HIF-1a, Rab7, MMP-14 and RECK in kidney tissues of mice were detected by western blot. (D–G) ß-actin was used for quantification. **P<0.01.

Fig 5: Defective endocytic machinery permits tau access to the cytosol in HEK-NGL cells(A) Levels of entry of 50 nM tau-HiBiT assemblies applied to HEK-NGL cells after RAB5A or RAB7A depletion. Cells were treated with siRNA or non-targeting control (NTC) siRNA for 72 h before assaying tau entry; n = 3, N = 3 independent experiments.(B) Western blot of cell lysates in (A), probing for RAB5, RAB7, and GAPDH.(C–F) Entry of tau-HiBiT assemblies into HEK-NGL cells after depletion of VPS13D (C and D) or VPS35 (E and F) and confirmation via western blotting following the same experimental procedure described in (A) and (B).(G) Fluorescence microscopy images of seeded tau-venus cells treated with NTC siRNA or VPS35-targeting siRNA for 72 h before addition of 250 nM exogenous tau assemblies in the absence of transfection reagents for another 72 h. Scale bars, 30 µm. White arrows indicate example fluorescent puncta.(H) Quantification of seeded aggregation from the tau-venus seeding assays in (G); n = 6.All error bars indicate mean ± SEM. **p < 0.01 by one-way ANOVA with Tukey's comparisons (C) or Kruskal-Wallis test with Dunn's comparisons (H). ****p < 0.0001 by one-way ANOVA with Tukey's comparisons (A and E).