Journal: eLife

Article Title: Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport

doi: 10.7554/eLife.81532

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti-Rab10 , Cell Signalling , Cat#8127; RRID: AB_10828219 , 1:200.

Techniques: Recombinant, shRNA, Clone Assay, Plasmid Preparation, Phospho-proteomics

Journal: Biology Open

Article Title: Rab35 promotes the recruitment of Rab8, Rab13 and Rab36 to recycling endosomes through MICAL-L1 during neurite outgrowth

doi: 10.1242/bio.20148771

Figure Lengend Snippet: (A) Active form-dependent interaction of Rab8A, Rab8B, Rab10, Rab13, Rab15, Rab35, and Rab36 with MICAL-L1-CC. Yeast two-hybrid assays were performed to investigate whether the Rabs indicated interacted with MICAL-L1-CC. Rab1A is a negative control that does not bind MICAL-L1 at all . (B) Colocalization of Rab8, Rab13, Rab35, and Rab36 with Arf6 in PC12 cells. After stimulating PC12 cells with NGF for 6 hr, the cells were fixed and stained with the anti-Rab antibodies indicated, anti-Arf6 antibody, and DAPI. The insets are magnified views of the boxed areas. Fluorescence intensity along the broken arrows is shown at the bottom. (C) Disappearance of MICAL-L1 signals from the perinuclear area of PC12 cells after depleting them of Rab35. PC12 cells were treated with siControl, siRab8 (siRab8A + siRab8B), siRab13, siRab35, or siRab36, and after stimulating the cells with NGF for 6 hr, the cells were fixed and stained with anti-MICAL-L1 antibody and DAPI. Scale bars: 10 µm. (D) Reduced expression of Rab8, Rab13, Rab35, and Rab36 in the cell treated with siRab8, siRab13, siRab35, and siRab36, respectively. Cell lysates of PC12 cells treated with siRNAs were immunoblotted with the anti-Rab antibodies indicated and anti-actin antibody. The asterisk indicates a non-specific band of the anti-Rab13 antibody. (E) Perinuclear MICAL-L1 signals (mean and SE; arbitrary units, a.u.) of siControl-treated, siRab8-treated, siRab13-treated, siRab35-treated, and siRab36-treated PC12 cells after stimulating the cells with NGF for 6 hr (n = 60 from 3 independent experiments).

Article Snippet: Anti-Arf6 mouse monoclonal antibody, anti-actin goat polyclonal antibody, anti-Myc tag mouse monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-MICAL-L1 mouse polyclonal antibody (Abnova, Taipei, Taiwan), anti-MICAL-L1 rabbit polyclonal antibody, anti-JIP4 rabbit polyclonal antibody (Abcam K. K., Tokyo, Japan), anti-Rab8 mouse monoclonal antibody (BD Biosciences, San Jose, CA), and anti-Rab10 rabbit monoclonal antibody (Cell Signaling Technology, Beverly, MA) were obtained commercially.

Techniques: Negative Control, Staining, Fluorescence, Expressing

Journal: Disease Models & Mechanisms

Article Title: Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease

doi: 10.1242/dmm.048017

Figure Lengend Snippet: Phosphorylation of bona fide LRRK2 substrates in LUHMES clones. (A) Representative WB results showing the phosphorylation of two LRRK2 bona fide substrates, LRRK2 Ser1292 and RAB10 Thr73, in naïve and clonal LUHMES cells (L10WT and L14GS). LUHMES cells were differentiated for up to 4 days, and levels of total and phosphorylated LRRK2 and RAB10 were analyzed each day. GAPDH was used to ensure equal loading. The protein corresponding molecular mass (in kDa) is indicated on the left side of the panel. (B) Quantification of pRAB10 levels. Results from six independent experiments. Error bars show mean±s.d.

Article Snippet: Control proteins were recombinant full-length human his-tagged RAB10 (Novus Biological, catalog no. NBP2-23392) and human full-length LRRK2 (ThermoFisher Scientific, catalog no. 15383806).

Techniques: Phospho-proteomics, Clone Assay

Journal: Disease Models & Mechanisms

Article Title: Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson's disease

doi: 10.1242/dmm.048017

Figure Lengend Snippet: LRRK2 kinase inhibitor MLI-2 effectively reduces phosphorylation of two bona fide LRRK2 substrates. (A) LUHMES cells were differentiated for 2 days and then treated with increasing concentrations of LRRK2 kinase inhibitor MLI-2 for 4 h. A potent reduction in the phosphorylation of LRRK2 Ser1292 and RAB10 Thr73 was observed. (B,C) Dose-response curves of the LRRK2 kinase inhibitor MLI-2 against LRRK2 pSer1292 and RAB10 pThr73 in WT and G2019S LUHMES cells. The protein corresponding molecular mass (in kDa) is indicated on the left side of the panel. (B) Half-maximal inhibitory concentration (IC 50 ) values, calculated for WT and G2019S LRRK2 by GraphPad Prism software on LRRK2 pSer1292, were found to be 21.2 pM (L10WT clone, filled circles) and 1.45 nM (L14GS clone, filled squares). (C) IC 50 values, calculated by GraphPad Prism software for WT and G2019S LRRK2 on RAB10 pThr73, were found to be 0.78 nM (L10WT clone, filled circles) and 2.31 nM (L14GS clone, filled squares). Results from three independent experiments.

Article Snippet: Control proteins were recombinant full-length human his-tagged RAB10 (Novus Biological, catalog no. NBP2-23392) and human full-length LRRK2 (ThermoFisher Scientific, catalog no. 15383806).

Techniques: Phospho-proteomics, Concentration Assay, Software

Journal: Communications Biology

Article Title: Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells

doi: 10.1038/s42003-021-02258-z

Figure Lengend Snippet: a The scheme summarizes the IP-MS hits in Ge518 shCtrl vs. shGal-3. b Histograms represent the fold change of normalized total spectra for significantly identified proteins by IP-MS analysis, in Ge518 shCtrl vs. shGal-3. c Immunoblot analysis of RAB10 immunoprecipitation from Ge518 shCtrl vs. shGal-3. Histograms represent the fold change of Gal-3 and RAB10 expression determined by densitometry analysis ( n = 3–4). WCL whole-cell lysate. d Immunoblots show expression of indicated proteins for Ge518 shCtrl or shGal-3. Histograms show the fold change of protein expression determined by densitometry analysis ( n = 3). e A cell-free binding assay shows direct binding between RAB10 and Gal-3 ( n = 4). f Immunoblots show expression of indicated proteins for Ge518 and Ge269 shCtrl vs. shRAB10. Histograms show the fold change of protein expression determined by densitometry analysis. g Effect of RAB10 knockdown on cell viability measured by CellTiter-Glo in Ge518 and Ge269. h Macropinocytosis uptake assay using TMR-dextran in Ge518 shCtrl vs. shRAB10. The histogram represents the fold change of macropinocytosis activity in Ge518 normalized to nuclei number ( n = 3). Data are represented as mean ± SEM (* p < 0.05, ** p < 0.01, and *** p < 0.001), two-way ANOVA, Dunnett’s multiple comparisons test. ns nonsignificant.

Article Snippet: Cells were infected with shRNAs for vector control (shCtrl, Open Biosystems), Gal-3 (Open Biosystems), RAB10 (Open Biosystems), PLX307 RAB10 (Addgene).

Techniques: Protein-Protein interactions, Western Blot, Immunoprecipitation, Expressing, Binding Assay, Knockdown, Activity Assay

Journal: Science advances

Article Title: Retromer promotes the lysosomal turnover of mtDNA.

doi: 10.1126/sciadv.adr6415

Figure Lengend Snippet: Fig. 6. The small GTPase RAB10 promotes mitochondrial fragmentation and mtDNA degradation in lysosomes. (A) Immunostaining of HeLa cells expressing the constitutive active protein RAB10Q68L-GFP labeled with α-VPS35. (B) Manders’ correlation coefficient between RAB10 and VPS35. (C and D) Confocal images of cells ex- pressing WT RAB10-GFP, constitutive active RAB10Q68L-GFP, dominant negative RAB10T23N-GFP, in the steady state, and (D) expressing TWNKK319E-mCherry, labeled with α-TOM20. (E) Quantification of the mitochondrial morphology in RAB10 expressing cells (n = 3, >20 cells per replicate). (F and G) Cells expressing RAB10Q68L-GFP and (G) TWNKK319E-mCherry labeled with α-LAMP1 and α-dsDNA. Arrows depict RAB10-LAMP1-dsDNA foci. (H) Manders’ correlation coefficient between RAB10-GFP and LAMP1 and LAMP1 and dsDNA (n = 3, 10 images per replicate). (I) RAB10-GFP coimmunoprecipitation in the steady state and cells expressing TWNKK319E-mCherry with the lysosomal protein LAMP1. P values were calculated using one-way ANOVA with Tukey correction for multiple comparisons. Scale bars, 10 μm. Data are presented as means ± SEM.

Article Snippet: RAB10 plasmids were obtained from Addgene (WT, Addgene #49472; Q68L Addgene #49544; and T23N Addgene #130885).

Techniques: Immunostaining, Expressing, Labeling, Dominant Negative Mutation

Journal: Science advances

Article Title: Retromer promotes the lysosomal turnover of mtDNA.

doi: 10.1126/sciadv.adr6415

Figure Lengend Snippet: Fig. 9. Proposed model for retromer function upon mtDNA stress. The retromer enhances mitochondrial fragmentation and mtDNA turnover. mtDNA ejection occurs in a BAX-dependent manner, targeting RAB10-VPS35-positive lysosomes, and independent of MDVs. Stimulation of these pathways restores mitochondrial function and mitigates defects associated with mtDNA damage in vivo.

Article Snippet: RAB10 plasmids were obtained from Addgene (WT, Addgene #49472; Q68L Addgene #49544; and T23N Addgene #130885).

Techniques: In Vivo