rtn4 nogoa  (Bio-Rad)

Journal: bioRxiv

Article Title: TBC1D24 regulates mitochondria and endoplasmic reticulum-mitochondria contact sites

doi: 10.1101/2024.09.19.613961

Figure Lengend Snippet: (A-B) Representative images showing the localisation of Flag-TBC1D24 (Flag, Green) in transfected primary fibroblasts, along with mitochondria (TOM20, Magenta)(A) or ER tubules (RTN4, Magenta)(B). (C-D) TBC1D24 mutations affect the structure of ER tubules. (C) Representative images ER tubules (RTN4, Red) and nuclei (Dapi, Blue) in control and patient fibroblasts. Scale bars 10 µm (D) Quantification of ER tubule phenotypes from images in (C). Cells with ER tubules similar to the patient fibroblast shown in (C) were considered as altered. Each point represents an individual experiment. Bars show the average ± SD. *** P<0.001. One-way ANOVA. (E-F) TBC1D24 mutations affect the structure of ER sheets. (E) Representative images ER tubules (CLIMP63, Red) and nuclei (Dapi, Blue) in control and patient fibroblasts. Scale bars 10 µm (F) Quantification of ER sheets phenotypes from images in (E). Cells with ER tubules similar to the patient fibroblasts shown in (E) were considered as altered. Each point represents an individual experiment. Bars show the average ± SD. * p<0.05, *** P<0.001. One-way ANOVA.

Article Snippet: The cells were then incubated for 1 hour at room temperature in blocking buffer with the following primary antibodies: TOM20 (Rb, Abcam, ab186735, 1:250), CLIM63 (Mo, ENZO, ENZ-ABS669, 1: 200), RTN4 (Rb, Bio-Rad, AHP1799, 1:200), FLAG M2 (Mo, Sigma-Aldrich, F1804, 1:200).

Techniques: Transfection, Control

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: Immunoblot analysis of proteinase K protection assay. HeLa cells were transfected with SCOTIN‐Myc (Total) and digitonin‐permeabilized. The supernatants (Cytosol) and pellet (PNSs) were separated by centrifugation. The PNSs were incubated with PBS or with proteinase K (+ PK) in the presence or absence of Triton X‐100 (+PK +Triton X). Representative high‐resolution microscopy images of Rab5 Q79L‐EGFP‐ and SCOTIN‐DsRed‐expressing HeLa cells. Magnified images show enlarged endosomes. I: SCOTIN on the endosomal membrane, II: SCOTIN on the endosomal membrane and luminal side. Scale bar: 10 μm. Scale bars in the magnified images: 5 μm. Plot analysis of SCOTIN relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on lines I and II from (B). The arrow indicates the boundary of the endosomal membrane. CLEM analysis of Rab5 Q79L‐EGFP‐overexpressing cells or Rab5 Q79L‐EGFP‐ and SCOTIN‐DsRed‐overexpressing cells. The arrow indicates dense accumulated ILVs. Distribution of endogenous SCOTIN (green), RTN4 (magenta), and CD63 (red) in SCOTIN‐FP 11 knock‐in cells. Representative Z projections of confocal images are shown. Arrows indicate endogenous SCOTIN with the RTN4. Arrowheads indicate the endogenous SCOTIN with the CD63. Left: Representative images of Rab5 Q79L‐mCherry‐expressing SCOTIN‐FP 11 knock‐in cells. Right: Plot analysis of SCOTIN‐FP 11 relative to the Rab5 Q79L signal shown. The graph indicates the fluorescence intensity of each channel on the lines. The arrow indicates the boundary of the endosomal membrane. Scale bars: 10 μm. Scale bars in the magnified images: 5 μm. Source data are available online for this figure.

Article Snippet: RTN4 (rabbit) , Bio‐Rad , Cat# AHP1799, RRID: AB_10612769.

Techniques: Western Blot, Transfection, Centrifugation, Incubation, Microscopy, Expressing, Membrane, Fluorescence, Knock-In

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: A Representative images from a PLA with RTN4 and Rab7 in WT and SCOTIN ‐KO cells. Only one primary antibody (anti‐RTN4 alone or anti‐Rab7 antibody alone) served as a negative control. The PLA color legends are inverted. All scale bars: 20 μm. B The number of PLA signals in each cell was quantified by particle analysis with ImageJ. C Schematic illustrating the SCOTIN domain. D Representative images of VAPA‐Rab7 PLA in WT and SCOTIN ‐KO cells. Cells were transfected with empty vector as a control, SCOTIN (FL)‐Myc, or SCOTIN (ΔPRD)‐Myc for 48 h. The PLA color legends are inverted. All scale bars: 30 μm. E The number of PLA signals in each cell was quantified by particle analysis with ImageJ. F–H Cellular localization of endogenous RTN4 (red) and CD63 (magenta) in (F) SCOTIN‐, (G) TMPRD‐, or (H) PRD‐Myc (green)‐reconstituted SCOTIN ‐KO cells. The graph indicates the fluorescence intensity of each channel on the lines. Data information: n : number of cells analyzed. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, **** P < 0.0001.

Article Snippet: RTN4 (rabbit) , Bio‐Rad , Cat# AHP1799, RRID: AB_10612769.

Techniques: Negative Control, Particle Size Analysis, Transfection, Plasmid Preparation, Control, Fluorescence, Two Tailed Test, Labeling

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: Left, representative images of Str‐Ii‐SCOTIN‐SBP‐EGFP along with the ER and late endosomes in SCOTIN ‐KO cells. Cells were stained with the ER tubular protein RTN4 (white) and the late endosomal marker CD63 (red). Untreated (−Biotin) or biotin‐treated (+Biotin 4 h). Arrows indicate EGFP signal with RTN4 and arrowheads indicate EGFP signal with CD63. Right, Schematic illustration of the relative localization of SCOTIN in Str‐Ii‐SCOTIN‐SBP‐EGFP‐expressing cells with or without biotin treatment. Scale bars: 20 μm. Representative images of VAPA‐Rab7 PLA signals taken from Str‐Ii‐SCOTIN‐SBP‐EGFP transfected SCOTIN ‐KO cells after untreated (−Biotin) or biotin treatment (+Biotin 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Representative images of Sec61β‐Emerald‐ or Sec61β‐PRD‐Emerald‐transfected SCOTIN ‐KO cells. Cells were stained with RTN4 (red). Arrowheads indicate the accumulated ER membrane. All scale bars: 20 μm. Representative images of Rab5 Q79L‐EGFP‐ and CD63‐ or CD63‐PRD‐Myc (red)‐transfected SCOTIN ‐KO cells. Arrowheads indicate the CD63‐ or CD63‐PRD‐Myc localization on the endosome membrane. All scale bars: 20 μm. Representative images of Sec61 or Sec61‐PRD‐Emerald with CD63 or CD63‐PRD‐Myc (red) cotransfected SCOTIN ‐KO cells. A colocalization image was obtained using the colocalization finder ImageJ Plugin. The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n : number of cells analyzed. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Source data are available online for this figure.

Article Snippet: RTN4 (rabbit) , Bio‐Rad , Cat# AHP1799, RRID: AB_10612769.

Techniques: Staining, Marker, Expressing, Transfection, Particle Size Analysis, Membrane, Two Tailed Test, Labeling

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: VLXT, VSL2, VL3, and IUPred2 assigned scores of disordered tendencies between 0 and 1 to the sequences are shown. Differential interference contrast (DIC) images of purified proteins (10 μM) in assembly buffer. Coomassie blue‐stained gels and immunoblot analysis showing the purified 12His‐PRDΔ150‐177 proteins used in the reconstitution study. Coomassie blue‐stained gels and immunoblot analysis of the cosedimentation assay. SUV‐bound 12His‐PRDΔ150‐177 were verified. PRDΔ150‐177‐SUVs and GUVs were mixed and subjected to confocal microscopy. Representative images from a PLA with RTN4 and Rab7 in SCOTIN ‐KO HeLa cells reconstituted with FL SCOTIN or SCOTIN(Δ150‐177)‐DsRed. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, * P < 0.05 and **** P < 0.0001. (F) and (G) are derived from three independent experiments. Immunoblot analysis of Ii‐SCOTIN(Δ150‐177)‐SBP‐EGFP‐transfected SCOTIN ‐KO HeLa cell lysates with or without biotin treatment. VAPA, Rab7, GFP, and α‐tubulin were immunoblotted.

Article Snippet: RTN4 (rabbit) , Bio‐Rad , Cat# AHP1799, RRID: AB_10612769.

Techniques: Purification, Staining, Western Blot, Confocal Microscopy, Transfection, Particle Size Analysis, Two Tailed Test, Labeling, Derivative Assay

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet: SUVs loaded with 12His‐PRDΔ150‐177 (PRDΔ150‐177‐SUV) were subjected to confocal microscopy. Representative confocal images from the liposome tethering assay with SUV‐ PRDΔ150‐177 and GUV‐PRD. HEK293 cells were transfected with SCOTIN‐GST along with empty (pcDNA3.1‐Myc), SCOTIN‐Myc, or SCOTIN(Δ150‐177)‐Myc plasmids for 48 h. SCOTIN‐GST was pulled down from total cell lysates using glutathione‐Sepharose beads, and the interacting proteins were analyzed by immunoblotting. Representative images from a PLA with VAPA and Rab7 in SCOTIN ‐KO HeLa cells reconstituted with FL SCOTIN, SCOTIN(ΔPRD), or SCOTIN(Δ150‐177)‐DsRed. The PLA color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. The number of PLA signals in each cell was quantified by particle analysis with ImageJ. Left, representative images of Str‐Ii‐SCOTIN(Δ150‐177)‐SBP‐EGFP along with the ER and late endosome in SCOTIN ‐KO cells. Cells were stained with the ER tubular protein RTN4 (white) and the late endosomal marker CD63 (red). Untreated (−Biotin) or biotin‐treated (+Bition 4 h). Arrows indicate EGFP signal with RTN4 and arrowheads indicate EGFP signal with CD63. Right, schematic illustration of the relative localization of SCOTIN(Δ150‐177) in Str‐Ii‐SCOTIN(Δ150‐177)‐SBP‐EGFP‐expressing cells with or without biotin treatment. Scale bars: 20 μm. Representative images of VAPA‐VAPA PLA signals taken from Str‐Ii‐SCOTIN(Δ150‐177)‐SBP‐EGFP transfected SCOTIN ‐KO cells after untreated (−Biotin) or biotin‐treated (+Bition 4 h). For the PLA signal, the color legends are inverted. Asterisks indicate transfected cells. All scale bars: 20 μm. The PLA signals in the indicated samples were quantified by particle analysis with ImageJ. Data information: n : number of cells analyzed. Data are shown as bars with dots and represent the means ± SEMs. P ‐values were determined using a two‐tailed unpaired t ‐test. Significant differences are labeled, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: RTN4 (rabbit) , Bio‐Rad , Cat# AHP1799, RRID: AB_10612769.

Techniques: Confocal Microscopy, Transfection, Western Blot, Particle Size Analysis, Staining, Marker, Expressing, Two Tailed Test, Labeling

Journal: EMBO Reports

Article Title: Homotypic SCOTIN assemblies form ER ‐endosome membrane contacts and regulate endosome dynamics

doi: 10.15252/embr.202256538

Figure Lengend Snippet:

Article Snippet: RTN4 (rabbit) , Bio‐Rad , Cat# AHP1799, RRID: AB_10612769.

Techniques: Recombinant, Fluorescence, Electron Microscopy, Knock-Out, Knock-In, Plasmid Preparation, Software, Microscopy