Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild‐type pGFP‐Rab8a (WT‐Rab8), constitutively active pGFP‐Rab8a (Q67L) (CA‐Rab8), or DN Rab8 dominant‐negative pGFP Rab8a (T22N), incubated in serum‐starved media for 12 h, and immunostained for ARL13B (red), GFP‐Rab8 (green), and DAPI (blue). Scale bar, 10 μm. Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST‐JCF1 (RBD). The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by ImageJ software. The amount of GTP‐Rab8 was normalized to the control level. Bar graph represents mean ± SD ( n = 3 experiments). * P < 0.05, Student's t ‐test. Cells were transfected as shown in (A), and cell lysates were incubated with purified GST‐JCF1 (RBD) fusion protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected as shown in (A), and cell lysates were incubated with purified GST protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blot with Rab8 antibody. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Dominant Negative Mutation, Incubation, Purification, Western Blot, Software, Control

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: RPE1 cells were transfected with siRNAs as indicated, followed by 24‐h incubation in serum‐starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB. Efficiency of IFT20 knockdown by Western blot. Cells were transfected with siRNAs as indicated, followed by incubation in serum‐starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD ( n = 3 experiments), and 250 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected as shown in (C), and immunostained for Rab8 and γ‐tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average ( n = 2 experiments). Cells were transfected with siRNAs as indicated, followed by transfection with Flag‐IFT20, incubated in serum‐starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag‐IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of ciliated cells with both the Flag‐IFT20 and ARL13B localized in the cilium. Data represent mean ± SD ( n = 3 experiments), and 150 Flag‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. Cells were transfected with siRNAs as indicated, followed by further transfection with CA‐Rab8, incubated in serum‐starvation media for 12 h, and immunostained for acetylated tubulin. Representative fluorescent images of GFP‐Rab8 Q67L (green), acetylated tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. Quantification of the percentage of GFP‐positive ciliated cells (only those cilia having both GFP‐Rab8 and acetylated tubulin on cilium were considered for quantification). Data represent mean ± SD ( n = 3 experiments), and 200 GFP‐positive cells were scored per condition per experiment, * P < 0.05, Student's t ‐test. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Transfection, Incubation, Fractionation, Western Blot, Marker, Membrane, Knockdown

Journal: EMBO Reports

Article Title: TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution

doi: 10.15252/embr.201948901

Figure Lengend Snippet: A Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells. B RPE1 cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for ARL13B (red) and γ‐tubulin (green). Scale bar, 10 μm. C Quantification of the percentage of ciliated cells shown in (B). Data represent mean ± SD ( n = 3 experiments), and 250 GFP‐positive cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. D Cells were transfected by siRNA as indicated followed by incubation in serum‐starvation media for 24 h, and immunostained for EHD1 (red) and γ‐tubulin (green). Scale bar, 10 μm. E Quantification of the percentage of cells with EHD1 in cilium or in the distal end of basal body as shown in (D). Data represent mean ± SD ( n = 3 experiments), and 150 cells were scored per condition per experiment; * P < 0.05, Student's t ‐test. F Cells were transfected by siRNAs as indicated, followed by incubation in serum‐starvation media for 24 h, and immunostained for IFT20 (red) and γ‐tubulin (green). Scale bar, 10 μm. G Quantification of the percentage of IFT20 fluorescent intensity at the centriole shown in (F). Data represent mean ± SD ( n = 3 experiments), and 150 were scored per condition per experiment; * P < 0.05, Student's t ‐test. H–J Cells were transfected by siRNAs as indicated, followed by transfection with GFP‐Rab8 WT, GFP‐Rab8 Q67L, or GFP‐Rab8 T22N, and further incubated in serum‐starvation media for 12 h. Cell lysate was subjected to immunoprecipitation with anti‐GFP antibody, followed by Western blot with antibody against GFP. Source data are available online for this figure.

Article Snippet: Human wild‐type pGFP‐Rab8A (Plasmid #24898), human constitutively active (Q67L) pGFP‐Rab8A (Plasmid #24900), and human dominant‐negative (T22N) pGFP‐Rab8A (Plasmid #24899) were obtained from Addgene.

Techniques: Western Blot, Transfection, Incubation, Immunoprecipitation

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Structure of pRab8a in Complex with the Phospho-Rab Binding Domain of RILPL2 (A) Heterotetrameric assembly of two pRab8a molecules bridged by a central α-helical dimer of the phospho-Rab binding domain of RILPL2 (129–165). The two chains of RILPL2 are in magenta and dark yellow. For pRab8a, switch 1 is shown in blue, switch 2 in red. (B) View of the complex down the 2-fold axis of the heterotetramer, 90° relative to orientation in (A). (C) Stick model of the RH2 domain of RILPL2. Rabs are stripped from the complex in this view, except for short segments of switch 1 and switch 2 (gray sticks). (D) Domain organization of RILPL1/2 showing the RH domains and their interacting partners. The sequence corresponds to RILPL2. (E) Simplified representation of the Rab:RILPL2 interface showing contacts between one molecule of Rab8a and the dimer of RILPL2. Polar interactions are indicated in dotted blue lines. Switch 1 (Sw1) and switch 2 are indicated. (F) Isothermal titration calorimetry analyses of the interactions between pRab8a and the phospho-Rab binding domain of RILPL2. Left, titration of RILPL2 (residues 129–165) into pRab8a(GTP). Right, titration of RILPL2 into Rab8a(GTP).

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Binding Assay, Sequencing, Isothermal Titration Calorimetry, Titration

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Mutational Analyses Reveal Hotspots of pRab8a:RILPL2 Interactions (A) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G], HA-Rab8a and WT or mutant RILPL2-GFP. At 48 h post transfection, cells were treated with ±500 nM MLi-2 for 90 min and then lysed. Upper panel, labeled IP:GFP: RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx western blot imaging system with the indicated antibodies at 0.5–1 μg/mL concentration. Lower panel, labeled input: 10 μg whole-cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments. (B) Same as A, but HEK293 cells were transiently transfected with WT or mutant HA-Rab8a as well as Flag-LRRK2[R1441G] and RILPL2-GFP WT. At 48 h post transfection, cells were treated with ±500 nM MLi-2 for 90 min and then lysed. RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and as in (A), immunoprecipitates and input were evaluated by immunoblotting with the indicated antibodies. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments. (C) Sequence alignment of the first α helix (α1) of the RILP family RH2 domains. Residues corresponding to the second α helix (α2) of RILP are not shown. Red circles are hotspots for the interactions where mutations severely reduce affinity between pRab8a and RILPL2. Blue circles indicate residues that are tolerant to mutations. The α-helical secondary structure above the alignment corresponds to RILPL2.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Transfection, Construct, Expressing, Mutagenesis, Labeling, Immunoprecipitation, Western Blot, Imaging, Concentration Assay, Sequencing

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Evidence that RILPL2 Binds to the GTP Bound Conformation of Phosphorylated Rab8a in Cells (A) Direct in vitro pull-downs were performed using purified His 6 -tagged RILPL2 (full length) as bait and untagged Rab8a as prey. Rab8a species were either non-phosphorylated (Rab8a) or phosphorylated (pRab8a). The GTP forms were stabilized via the Q67L mutation in switch 2. The GDP form of Rab8a was prepared by in vitro exchange using wild-type (WT) Rab8a before the phosphorylation reaction to generate pRab8a(GDP). Protein concentrations were 10 μM for bait and prey, inputs are 2 μg; n ≥ 3, Coomassie stain for visualization. Dotted lines emphasize that only pRab8a(GTP) binds to RILPL2. (B) HEK293 cells were transiently transfected with constructs expressing the indicated components. 24 h post transfection, cells were treated with ±100 nM MLi-2 for 90 min and then lysed. Upper panel, labeled IP:GFP: RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx western blot imaging system with the indicated antibodies at 0.5–1 μg/mL concentration. Lower panel, labeled input: 10 μg whole-cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: In Vitro, Purification, Mutagenesis, Phospho-proteomics, Staining, Transfection, Construct, Expressing, Labeling, Immunoprecipitation, Western Blot, Imaging, Concentration Assay

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: MyoVa Interactions with the RH1 Domain Enhance the Affinity of RILPL2 to pRab8a (A) Pull-downs of (p)Rab8a and RILPL2 in the presence of MyoVa(GTD). Input proteins are in the upper left panel, while duplicate pull-downs are shown to the right. Phosphorylated Rab8a (pRab8a) is highlighted in the pull-down lanes with red (+) labels. Bait and prey proteins were used at 2.5 μM. (B) Control experiment showing that no interactions are observed between His 6 -tagged MyoVa and pRab8a/Rab8a. (C) Quantification of densitometry readings of pRab8a pull-downs from three independent experiments (p < 0.005). (D) Modeling of full-length RILPL2 using ribbons and electrostatic surfaces. The RH1 domain of mouse RILPL2 was connected to the RH2 domain of human RILPL2. Residue numbers correspond to the human protein.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Control, Residue

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Structural Comparisons of Rab:Effector Complexes (A) Structure of Rab7 in complex with the Rab binding domain of RILP. (B) Superposition of RILPL2 onto a single binding interface of Rab7:RILP, showing conservation of the α-helical coiled coil. The figure is rotated 90° along the horizontal axis, relative to (A). (C) Structure of Rab8a in complex with OCRL1. The dashed circle denotes the region that sterically clashes with pT72 of pRab8a. (D) Close-up view of the switch 2 region denoted by the dashed circle. Here, pRab8a (red) from the complex with RILPL2 is superimposed onto the structure of Rab8a (gray) in complex with OCRL1. The distances between the methyl groups from the β-branched sidechains of pT72 and Ile71 are shown to highlight the steric clashes.

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Binding Assay

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet: Model for the Control of Rab8a Functions by LRRK2 Rab29 recruits LRRK2 to membranes and Rab8a is subsequently phosphorylated by LRRK2. RILPL2 is then recruited to membranes by pRab8a via the X-cap. RILPL2 is an adaptor that links pRab8a to the GTD of MyoVa. The structure of the mouse complex of RILPL2 with myosin was used to generate this figure (PDB: 4kp3 ; Wei et al., 2013 ).

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Control

Journal: Structure(London, England:1993)

Article Title: Structural Basis for Rab8a Recruitment of RILPL2 via LRRK2 Phosphorylation of Switch 2

doi: 10.1016/j.str.2020.01.005

Figure Lengend Snippet:

Article Snippet: The cDNA for Rab8a (residues 1-181, Q67L) lacking the flexible C-terminal tail was ordered from Genscript in a codon-optimized form to enable E . coli expression.

Techniques: Recombinant, Residue, Mutagenesis, Software

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: PPM1H binds nonphosphorylated Rab8A and Rab10 but not Rab12 via Leu66. Microscale thermophoresis of mNeon PPM1H and mNeon L66R PPM1H with Rab10 Q68L ( A and D ), Rab8A Q67L ( B , E ), or Rab12 Q101L ( C and F ). Purified Rab proteins were serially diluted, and mNeon PPM1H or mNeon L66R PPM1H was added (final concentration of 100 nM). Graphs show the mean ± SD from three independent measurements, each using different protein preparations. Values are summarized in .

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Microscale Thermophoresis, Purification, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: Binding properties of Rab8A and thiophosphorylated Rab8A to PPM1H. A and B , thiophosphorylated Rab8A Q67L does not rely on L66 for PPM1H binding. C and D , thio-phosphorylated Q67L Rab8A but not nonphosphorylated Rab8A binds much less tightly to a PPM1H FLAP domain mutated PPM1H R338A. E and F , GTP-bound WT Rab8A binds more strongly than GDP-bound WT Rab8A to PPM1H. Various Rab8A or pRab8A proteins were serially diluted and incubated with 100 nM mNeon PPM1H variants as in . Graphs represent mean ± SD from three independent experiments using separate protein preparations. pRab, phosphoRab.

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Binding Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: Rab8A associates with liposome-bound PPM1H. Sucrose gradient coflotation of His-Rab8A Q67L (full length, nonprenylated) with mNeon PPM1H ( A , B ) or mNeon L66R PPM1H ( C , D ) in the presence and absence of 50 nm liposomes. The distribution of PPM1H and Rab8A across the gradient was determined by immunoblot; fractions were collected from the top . Quantification of three independent experiments is shown (±SD).

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Liposomes, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: PPM1H’s N-terminal residues contribute to Rab8A and Rab10 binding. Microscale thermophoresis of mNeon Δ37 PPM1H with His Rab8A Q67L ( A ) or Rab10 Q68L ( B ). C , sucrose gradient coflotation of His Rab8A Q67L with mNeon Δ37 PPM1H in the presence and absence of 50 nm diameter NTA(Ni) containing liposomes. D , quantification of three independent experiments is shown (±SD). Ni, nickel; NTA, nitrilotriacetic acid.

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Binding Assay, Microscale Thermophoresis, Liposomes

Journal: The Journal of Biological Chemistry

Article Title: Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease

doi: 10.1016/j.jbc.2025.110679

Figure Lengend Snippet: Rab8A, but not Rab12, inhibits PPM1H activity. A , anti-pRab10 immunoblot analysis of dephosphorylation by PPM1H ( upper gel ) or L66R PPM1H ( lower gel ). B and C , data were plotted as PPM1H activity, with maximum activity equal to the amount of pRab10 dephosphorylation seen in the absence of non-pRab inhibitor (lanes 3 and 4 of each gel) compared with reactions lacking PPM1H (lanes 1 and 2). D , rate of pRab10 phosphatase activity (monitored at t = 0, 8, 15, and 30 min) for mNeon-PPM1H, mNeon-L66R PPM1H, and mNeon Δ37 PPM1H. E , quantification of pRab10 intensity from ( D ), with maximum intensity detected at 0 min normalized to a value of 1. Error bars represent SD from three independent experiments; representative gels are shown. Black line , mNeon L66R PPM1H activity; red line , mNeon PPM1H activity; and blue line , mNeon Δ37 PPM1H activity. pRab, phosphoRab.

Article Snippet: Recombinant DNA , pET14b His Rab8A (Q67L) full length , Addgene , 186014; RRID: Addgene_1860014 , Reuse , .

Techniques: Activity Assay, Western Blot, De-Phosphorylation Assay

Journal: eLife

Article Title: A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

doi: 10.7554/eLife.79771

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pGEB GST-Rab8A-Q67L , Addgene RRID: Addgene_86079 , 86079 , Human.

Techniques: Labeling, Recombinant, Software