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: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a , b Knockdown of RAB8 expression by RAB8 siRNA in H1792 ( a ) and A549 ( b ) cells in the presence or absence of PEM at 5.0 μM for 36 h. The surface expression of TNFRSF10B was confirmed by flow cytometry analyses. c Overexpression of RAB8 in H1299 cells in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against RAB8, TNFRSF10B, CASP8, CASP3, PARP1 and ACTB. d Knockdown of RAB8 expression by RAB8 siRNA in H1792 cells in the presence or absence of PEM at 5.0 μM for 36 h. Cell lysates were analyzed by Western blotting with antibodies against RAB8, TNFRSF10B, CASP8, CASP3, PARP1 and ACTB.

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Expressing, Flow Cytometry, Over Expression, Western Blot

Journal: Cell Death & Disease

Article Title: YIPF2 promotes chemotherapeutic agent-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in non-small cell lung cancer cells

doi: 10.1038/s41419-020-2436-x

Figure Lengend Snippet: a H1299 cells were transfected with pcDNA3.1 or pcDNA3.1-Flag-TNFRSF10B (short isoform) plasmids. Then the co-IP assays were carried out with Flag antibody and the co-eluted proteins were detected by western blot assays with Flag and YIPF2 antibodies. b , c H1792 cells were transfected with pEGFP-N1 or pEGFP-N1-YIPF2 plasmids. Then the co-IP assays were carried out with GFP antibody and the co-eluted proteins were detected by western blot assays with GFP, TNFRSF10B ( b ) and RAB8 ( c ) antibodies. d H1792 cells were transfected with pEGFP-N1 or pEGFP-N1-RAB8 plasmids. Then the co-IP assays were carried out with GFP antibody and the co-eluted proteins were detected by western blot assays with GFP, and TNFRSF10B antibodies. e Cell lysates were prepared from H1792 cells transiently co-expressing pEGFP-N1-RAB8 and pcDNA3.1-YIPF2. The co-IP assays were carried out with GFP antibody and the co-eluted proteins were detected by western blot assays with YIPF2, GFP and TNFRSF10B antibodies. f Schematic illustration showing that YIPF2 promotes PEM-mediated apoptosis via enhancing TNFRSF10B recycling to plasma membrane in NSCLC cells.

Article Snippet: The primary antibodies used in western blot assays and Immunoprecipitation were as follows: anti-YIPF2 (Cat. no. HPA019902; Sigma-Aldrich), ACTB (Cat. no. A1978; Sigma-Aldrich), CASP8 (Cat. no. 9746S; CST), CASP3 (Cat. no. NB100-56708; Novus Biologicals), PARP1 (Cat. no. 9542S; CST), TNFRSF10B (Cat. no. 2019; ProSci Incorporated), RAB8 (Cat. no. 6975S; CST), Flag (Cat. no. F7425, F1804; Sigma-Aldrich) and GFP (Cat. no. G1544, Sigma-Aldrich; Cat. no. sc-9996, Santa Cruz).

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing

Journal: PLOS ONE

Article Title: Rare CIDEC coding variants enriched in age-related macular degeneration patients with small low-luminance deficit cause lipid droplet and fat storage defects

doi: 10.1371/journal.pone.0280484

Figure Lengend Snippet: (A) 3xflag-tagged CIDEC wild-type (WT) was co-transfected with the indicated GFP-tagged CIDEC variants in HEK 293T cells. GFP alone was used as negative control. 3xflag-tagged CIDEC WT was immuno-precipitated (IP) using anti-Flag and pulled-down proteins were immuno-blotted (IB) with anti-GFP and anti-Flag. Total cell lysate was immunoblotted with anti-GFP to control for CIDEC-GFP expression levels. (B-E) HEK 293T cells were co-transfected with 3xFlag-CIDEC WT, E186X or AMD variants, and either PLIN1-mCherry (B), AS160-GFP (C) or RAB8A-mCherry (D). After immunoprecipitation (IP) of the 3xFlag-CIDEC, pulled-down proteins were probed with anti-mCherry or anti-GFP, and anti-Flag. Co-transfection with mCherry or GFP alone was used as negative controls. Total cell lysates were immunoblotted (IB) with anti-mCherry or anti-GFP to control for PLIN1, AS160 and RAB8A expression levels. (E) Representative fluorescence images of 3T3-L1 pre-adipocytes lipid droplets containing CIDEC-GFP wild-type (WT) or variants and RAB8A-mCherry. Scale bar: 2 μm.

Article Snippet: GFP- and mCherry-tagged plasmids were used to express human PLIN1, AS160, and RAB8A (Genecopoeia, Inc.).

Techniques: Transfection, Negative Control, Control, Expressing, Immunoprecipitation, Cotransfection, Fluorescence

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Primer sequences to clone hSVCT1 and Rab8a and real-time PCR primers

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Real-time Polymerase Chain Reaction

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2, HT-29, NCM460 and HuTU-80 cells were transiently co-transfected with hSVCT1-YEP and DsRed-Rab8a constructs. Data are from n > 6-10 transfected cells. Scale bar is 10 μm

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Transfection, Construct

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) 14C-AA uptake (32 μM) was performed on Rab8a siRNA treated hSVCT1 expressing Caco-2 cells. Data are mean ± SE of at least three separate experiments performed on different batches of cells on separate occasions. *p < 0.02. B) Real-time PCR was performed using gene specific primers for Rab8a and β-actin from total RNA isolated from Rab8a siRNA and control siRNA (scrambled) treated Caco-2 cells. Data are mean ± SE of at least three independent experiments. *p < 0.01. C, Top, Western blot analysis was performed on cell extract (60 μg) isolated from control (left) and Rab8a siRNAs treated Caco-2 cells (right). Blots were incubated with rabbit polyclonal anti-human Rab8a specific antibodies (top) along with β-actin antibodies (bottom). Bottom, densitometric quantification of the immunoreactive bands. Data are mean ± SE of at least three separate sample preparations. *p < 0.01. D) Carrier-mediated 14C-AA uptake (32 μM) by Rab8a KO mice jejunal portion was performed as described in “Materials and Methods”. Values are mean ± SE of at least three separate uptake determinations from multiple sets of mice. *p < 0.01. E) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type (litter-mate) mice jejunal mucosa using mouse Rab8a and β-actin gene-specific primers. Data are mean ± SE of at least three separate samples from three different mice. *p < 0.01. F, Top) Western blot analysis was performed on Rab8a KO (right) and wild-type litter-mate (left) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit polyclonal anti-mouse Rab8a antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three sets of samples from three different mice.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) Real-time PCR was performed using primers for hSVCT1, hSVCT2 and β-actin on total RNA isolated from control (scrambled) and Rab8a siRNAs treated Caco-2 cells. Data are mean ± SE of multiple experiments performed on different batches of cells. B, Top, western blot analysis was performed on cell extract (60 μg) isolated from control and Rab8a siRNAs treated Caco-2 cells. Blots were incubated with rabbit anti-human hSVCT1 specific antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of multiple experiments performed on separately isolated samples.*p < 0.01. C) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type litter-mate mice jejunal mucosa using mouse SVCT1, SVCT2 and β-actin primers. Data are mean ± SE of at least three separate samples from three different mice. D, Top, western blot analysis was performed on Rab8a KO and wild-type (litter-mate) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit anti-mouse SVCT1 antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three separate samples from three mice. *p < 0.02.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2 cells were transiently transfected with Rab8a siRNA or control siRNA. Forty eight hours after transfection cells were processed for biotinylation. Equal amount of protein was loaded onto pre-made 4-12% mini-gel and western blot was performed using anti-hSVCT1 antibody. The level of cell surface expression was normalized relative to the total amount of cellular hSVCT1 protein. Densitometric values are from mean ± SE of four independent experiments. Inset shows representative western blot images. *p < 0.01.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Transfection, Control, Western Blot, Expressing

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Hu-Tu-80 cells were co-transfected with hSVCT1-YEP, LAMP1-RFP, control or Rab8a siRNAs. Live cell confocal imaging (laterl sections-xy) was performed after 48 h of transfection. Data are from n > 6-10 transfected cells and representative images were shown. Scale bar is 10 μm.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Transfection, Control, Imaging

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