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rabbit anti gfp  (Millipore)

 
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    Millipore rabbit anti gfp
    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: <t>plasma</t> <t>membranes</t> labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with <t>GFP</t> at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.
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    Images

    1) Product Images from "A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts"

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202311137

    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.
    Figure Legend Snippet: Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

    Techniques Used: Labeling, Membrane, Fluorescence, Expressing

    TMEM24, but not other ER/PM tethers, concentrate as cell–cell junctions. HEK293 cells. (A and B) TMEM24 expressed in one cell generates enlarged ER/PM junctions that are not mirrored by eGFP-ESyt2-positive or eGFP-JPH4-positive junctions in directly adjacent cells. (C) The accumulation of TMEM24-mCherry expressed in one cell at a cell-adjacent ER/PM junction is mirrored by the accumulation of TMEM24-GFP expressed in the adjacent cell (see also ). (D) ER/PM junctions induced by mCherry-ESyt2 and GFP-ESyt2-expressed in two adjacent cells respectively, do not mirror one another across the cell–cell interface. (E) mCherry-JPH4 and GFP-JPH4 do not robustly mirror one another across the cell–cell interface although junctions could be found that seemed to be symmetrically opposed. (F) eGFP-Esyt2 colocalizes with TMEM24 at ER/PM junctions, but is excluded from the cell adjacent junction where TMEM24-GFP is selectively enriched. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (G) Kv2.1(S601,607D)-GFP, a Kv2.1 construct that binds constitutively to the ER protien VAP colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (H) STIM1(D76A)-GFP, a STIM1 construct that constitutively binds the PM, colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right.
    Figure Legend Snippet: TMEM24, but not other ER/PM tethers, concentrate as cell–cell junctions. HEK293 cells. (A and B) TMEM24 expressed in one cell generates enlarged ER/PM junctions that are not mirrored by eGFP-ESyt2-positive or eGFP-JPH4-positive junctions in directly adjacent cells. (C) The accumulation of TMEM24-mCherry expressed in one cell at a cell-adjacent ER/PM junction is mirrored by the accumulation of TMEM24-GFP expressed in the adjacent cell (see also ). (D) ER/PM junctions induced by mCherry-ESyt2 and GFP-ESyt2-expressed in two adjacent cells respectively, do not mirror one another across the cell–cell interface. (E) mCherry-JPH4 and GFP-JPH4 do not robustly mirror one another across the cell–cell interface although junctions could be found that seemed to be symmetrically opposed. (F) eGFP-Esyt2 colocalizes with TMEM24 at ER/PM junctions, but is excluded from the cell adjacent junction where TMEM24-GFP is selectively enriched. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (G) Kv2.1(S601,607D)-GFP, a Kv2.1 construct that binds constitutively to the ER protien VAP colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (H) STIM1(D76A)-GFP, a STIM1 construct that constitutively binds the PM, colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right.

    Techniques Used: Fluorescence, Construct, Membrane

    Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .
    Figure Legend Snippet: Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

    Techniques Used: Affinity Purification, Western Blot, Construct

    A TMEM24-4.1-SynCAM1 complex at sites of cell – cell contact. (A) Schematic of the SynCAM 1(363)-GFP construct. (B) Confocal images of SynCAM1(363)-eGFP expressed in HEK293 cells. The upper panel is a slice at mid z level while the lower panel is shown as a basal surface. Representative example of n = 22 cells. (C) Coexpression of TMEM24-mCherry and SynCAM1(363)-eGFP in HEK293 cells. Images are of a mid-level z-slice (above) or a basal surface (below) of the cell. Plasma membrane labeled with CellBrite 650. Representative example of n = 35 cells. (D) . Coexpression of JPH4-mCherry and SynCAM1(363)-eGFP in HEK293 cells. The plasma membrane was labeled with CellBrite 650. Representative example of n = 28 cells (E) Pearson’s correlation R values of comparisons of the SynCAM1(363)-eGFP fluorescence with the fluorescence of either TMEM24-mCherry or mCherry-JPH4 (P = 6.76E-11, TMEM24 n = 24, JPH4 n = 30). (F) Expression of SynCAM1(363)-eGFP, mCherry-4.1G, and TMEM24-Halo labeled with JF646 HaloTag ligand in HEK293 cells. All three proteins colocalize at ER/PM junctions. Representative example of n = 74 cells. ( G ) Model of the TMEM24-4.1-SynCAM 1 complex. Diagonal lines indicate regions of the micrographs occupied by cells. Scale bars = 5 μm.
    Figure Legend Snippet: A TMEM24-4.1-SynCAM1 complex at sites of cell – cell contact. (A) Schematic of the SynCAM 1(363)-GFP construct. (B) Confocal images of SynCAM1(363)-eGFP expressed in HEK293 cells. The upper panel is a slice at mid z level while the lower panel is shown as a basal surface. Representative example of n = 22 cells. (C) Coexpression of TMEM24-mCherry and SynCAM1(363)-eGFP in HEK293 cells. Images are of a mid-level z-slice (above) or a basal surface (below) of the cell. Plasma membrane labeled with CellBrite 650. Representative example of n = 35 cells. (D) . Coexpression of JPH4-mCherry and SynCAM1(363)-eGFP in HEK293 cells. The plasma membrane was labeled with CellBrite 650. Representative example of n = 28 cells (E) Pearson’s correlation R values of comparisons of the SynCAM1(363)-eGFP fluorescence with the fluorescence of either TMEM24-mCherry or mCherry-JPH4 (P = 6.76E-11, TMEM24 n = 24, JPH4 n = 30). (F) Expression of SynCAM1(363)-eGFP, mCherry-4.1G, and TMEM24-Halo labeled with JF646 HaloTag ligand in HEK293 cells. All three proteins colocalize at ER/PM junctions. Representative example of n = 74 cells. ( G ) Model of the TMEM24-4.1-SynCAM 1 complex. Diagonal lines indicate regions of the micrographs occupied by cells. Scale bars = 5 μm.

    Techniques Used: Construct, Membrane, Labeling, Fluorescence, Expressing



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    Revvity Signals rabbit polyclonal anti gfp antibody
    Intracellular AIMP2 accumulation leads to AIMP2 secretion and intercellular transmission. ( A ) Representative immunofluorescence images of endothelial marker CD31 and AIMP2 in the cortical brain subregions from two- and six-month-old AIMP2 transgenic mice and age-matched littermate controls. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar = 10 μm. ( B ) Quantification of AIMP2 signal intensities in CD31-positive brain endothelium in the indicated mouse groups ( n = 5 in two-month-age group, n = 5 in control six-month-age group, and n = 8 in AIMP2 Tg six-month-age group). ( C ) Anti-AIMP2 dot blot assessment of <t>GFP-AIMP2</t> protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( D ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with GFP or GFP-AIMP2 based on the dot blot result in the panel C ( n = 3 separate experiments per group). ( E <t>)</t> <t>Anti-GFP</t> dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( F ) Quantification of <t>relative</t> <t>anti-GFP</t> dot blot optical densities for experimental groups in the panel E ( n = 3 separate experiments per group). ( G ) Dot blot assessment of AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with GFP-AIMP2 construct (0, 1, 2 μg). Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( H ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with the indicated combination of GFP and GFP-AIMP2 ( n = 3 separate experiments per group). ( I ) Representative immunofluorescence images showing GFP-AIMP2 uptake into HUVECs. HUVECs were treated with conditioned media (48 h) from GFP or GFP-AIMP2 transfected SH-SY5Y cells. Scale bar = 50 μm. ( J ) Percentage GFP-positive HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). ( K ) Quantification of GFP-AIMP2 immunofluorescence signals in HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). Quantitative data are expressed as the mean ± SEM, and statistical significance was determined by ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. ns, non-significant
    Rabbit Polyclonal Anti Gfp Antibody, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti gfp  (MBL Life science)
    86
    MBL Life science rabbit anti gfp
    REEP5 interacts <t>with</t> <t>MFN1/2</t> through C-terminal cytosolic domains. All co-immunoprecipitation (Co-IP) experiments were done in HEK293T cells. (A and B) Co-IP analysis of Myc-tagged REEP 1–6 with Flag-MFN1/2. (C) Co-IP analysis of endogenous MFN1/2 with REEPs and Rtns. (D) Co-IP analysis of Flag-MFN2 wild type or Flag-MFN2 ActA with REEP5-Myc. (E) Schematic diagram of human REEP5 as previously reported ( ; ), MFN1 according to UniProt, and MFN2 according to . HR, coiled-coiled heptad repeat domain; TM, transmembrane; APH, amphipathic helix; cytL, cytosolic loop; CTH, C-terminal helix. (F and G) Co-IP analysis of mEmerald-tagged REEP5 FL or truncation mutants with Flag-MFN1/2, and <t>GFP-tagged</t> MFN1/2 FL or truncation mutants with REEP5-Myc. FL, full length. Numbers indicate amino acids in each construct. (H) Purified His-REEP5, GST-MFN1/2 C-terminal cytosolic domains, and GST were analyzed by Coomassie staining. (I) GST pull-down of GST-MFN1/2 C-terminal cytosolic domains with His-REEP5. Source data are available for this figure: .
    Rabbit Anti Gfp, supplied by MBL Life science, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    FAM177A1 is a protein neighbor of VPS13B in the Golgi complex. (A) Alphafold2 prediction of human FAM177A1 structure. (B) COS7 cells expressing VPS13B^Halo, FAM177A1-GFP, and GalT-RFP showing that FAM177A1 is localized at the Golgi complex. (C) Western blot analysis of soluble cytosolic fraction and total membrane faction of HeLa cells expressing FAM177A1-GFP. GAPDH was used as a soluble control, Stx6 as a membrane control protein. (D) Top panel: Co-expression of FAM177A1-helix1-GFP and GalT-RFP in COS7 cells. Bottom panel: Co-expression of FAM177A1-Δhelix 1-GFP and GalT-RFP in COS7 cells. Scale bar = 10 µm. (E) FLASH-PAINT performed in HeLa cells expressing VPS13B^GFP and FAM177A1-Halo and immunolabeled with anti-GFP, anti-halo, and anti-GM130 antibodies. (F) Median distances between super-resolved signals of different Golgi complex targets. Only signals closer than 500 nm to each other were considered. (G) Snapshots of COS7 cells expressing FAM177A1-SNAP, ZFPL1-GFP, and GalT-RFP treated with water for 10 min. Scale bar = 5 µm. (H) Snapshots of HeLa cells expressing FAM177A1 GFP and VPS13B^Halo before and after BFA (5 µg/ml for 50 min) treatment. Scale bar = 10 µm. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: VPS13B is localized at the interface between Golgi cisternae and is a functional partner of FAM177A1

    doi: 10.1083/jcb.202311189

    Figure Lengend Snippet: FAM177A1 is a protein neighbor of VPS13B in the Golgi complex. (A) Alphafold2 prediction of human FAM177A1 structure. (B) COS7 cells expressing VPS13B^Halo, FAM177A1-GFP, and GalT-RFP showing that FAM177A1 is localized at the Golgi complex. (C) Western blot analysis of soluble cytosolic fraction and total membrane faction of HeLa cells expressing FAM177A1-GFP. GAPDH was used as a soluble control, Stx6 as a membrane control protein. (D) Top panel: Co-expression of FAM177A1-helix1-GFP and GalT-RFP in COS7 cells. Bottom panel: Co-expression of FAM177A1-Δhelix 1-GFP and GalT-RFP in COS7 cells. Scale bar = 10 µm. (E) FLASH-PAINT performed in HeLa cells expressing VPS13B^GFP and FAM177A1-Halo and immunolabeled with anti-GFP, anti-halo, and anti-GM130 antibodies. (F) Median distances between super-resolved signals of different Golgi complex targets. Only signals closer than 500 nm to each other were considered. (G) Snapshots of COS7 cells expressing FAM177A1-SNAP, ZFPL1-GFP, and GalT-RFP treated with water for 10 min. Scale bar = 5 µm. (H) Snapshots of HeLa cells expressing FAM177A1 GFP and VPS13B^Halo before and after BFA (5 µg/ml for 50 min) treatment. Scale bar = 10 µm. Source data are available for this figure: .

    Article Snippet: Primary antibodies used were as follows: rabbit VPS13B (24505-1-AP; RRID:AB_2879579; IB (immuno-blot) 1:250; Proteintech); rabbit FAM177A1 (A303-366A; RRID:AB_10952864; IB 1:1,000; Bethyl Laboraties); mouse GM130 (610822; RRID:AB_398141; IF (immuno-fluorescence) 1:500; BD Bioscience); rabbit GOLGA2/GM130 (ab30637; RRID:AB_732675 IF 1:300; Abcam and 11308-1-AP; RRID:AB_2115327; Proteintech); rabbit TGN46 (ab50595; RRID:AB_2203289; IF 1:200; Abcam and 10598-1-AP; RRID:AB_2113473; Proteintech); mouse GAPDH (40-1246; IB 1:10,000; Proteus); mouse α-Tubulin (T5168; RRID:AB_477579; IB 1:10,000; Sigma-Aldrich); rabbit GFP (A-11122, RRID: AB_221569, IF 1:250; IB 1:1,000; Invitrogen); rabbit Stx6 (110 062; RRID:AB_887854; IB 1:1,000; Synaptic systems); GRASP 65 (ab174834; Abcam); Giantin (HPA011555; RRID:AB_1079010; Sigma-Aldrich); Golgin 97 (HPA044329; RRID:AB_2678897; Atlas antibodies); sheep anti-GALNT2 (AF7507; Novus); COPI (CMIA10) were customary made in the Rothman lab.

    Techniques: Expressing, Western Blot, Membrane, Control, Immunolabeling

    Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    doi: 10.1083/jcb.202311137

    Figure Lengend Snippet: Large TMEM24-positive ER/PM junctions at cell–cell interfaces. (A) Left: Spinning disk confocal images of TMEM24-mCherry, C2CD2-eGFP, and JPH4-eGFP in HEK293 cells. Center: plasma membranes labeled with CellBrite 650 dye. Faint diagonal lines have been added to indicate regions of the micrographs occupied by the cells. Right: ER/PM junctions from the left fields with cell-adjacent ER/PM junctions are outlined in red. (B) ROIs were drawn around regions of the plasma membrane touching an adjacent cell or facing open space and the mean fluorescence intensity of the indicated proteins within those regions was measured. An enrichment ratio was calculated for each cell by dividing the mean fluorescent intensity at adjacent regions by the mean non-adjacent fluorescent intensities. Student’s t test; P values of P = 0.0010 for TMEM24 and P = 7.12902E-07 for C2CD2. (C and D) Cells expressing TMEM24-eGFP and TMEM24-mCherry were coplated as illustrated by the diagram. (C) TMEM24 expressed in adjacent cells forms symmetrical ER/PM junctions. (D) Gaps in the TMEM24 signal (indicated by arrowheads) in one cell are mirrored by gaps in the signal of the adjacent cell. (E) Diagram illustrating TMEM24 behavior in adjacent cells expressing TMEM24. (F) TMEM24 tagged with GFP at the endogenous locus in IMR32 cells localizes preferentially to the cell–cell interface. (G) Quantification of the enrichment ratio for mean fluorescence of TMEM24 tagged at the endogenous locus (endoTMEM24) at regions of IMR32 cells that face adjacent cells or empty space in the dish. As endoTMEM24 signal may stem from both adjacent cells, the observed fluorescence at the adjacent region has been halved and compared against non-adjacent regions (P = 0.000000003, n = 24 cells). (H) Differentiated IMR32 cells show that endoTMEM24 fluorescence is preferentially localized at sites of cell–cell contact as in the undifferentiated IMR32 cells. In F and H, the plasma membrane was labeled with CellBrite 650 dye. Diagonal lines indicate regions of the micrographs occupied by cells to clearly differentiate these regions from empty spaces. Scale bars = 5 μm.

    Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

    Techniques: Labeling, Membrane, Fluorescence, Expressing

    TMEM24, but not other ER/PM tethers, concentrate as cell–cell junctions. HEK293 cells. (A and B) TMEM24 expressed in one cell generates enlarged ER/PM junctions that are not mirrored by eGFP-ESyt2-positive or eGFP-JPH4-positive junctions in directly adjacent cells. (C) The accumulation of TMEM24-mCherry expressed in one cell at a cell-adjacent ER/PM junction is mirrored by the accumulation of TMEM24-GFP expressed in the adjacent cell (see also ). (D) ER/PM junctions induced by mCherry-ESyt2 and GFP-ESyt2-expressed in two adjacent cells respectively, do not mirror one another across the cell–cell interface. (E) mCherry-JPH4 and GFP-JPH4 do not robustly mirror one another across the cell–cell interface although junctions could be found that seemed to be symmetrically opposed. (F) eGFP-Esyt2 colocalizes with TMEM24 at ER/PM junctions, but is excluded from the cell adjacent junction where TMEM24-GFP is selectively enriched. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (G) Kv2.1(S601,607D)-GFP, a Kv2.1 construct that binds constitutively to the ER protien VAP colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (H) STIM1(D76A)-GFP, a STIM1 construct that constitutively binds the PM, colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right.

    Journal: The Journal of Cell Biology

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    doi: 10.1083/jcb.202311137

    Figure Lengend Snippet: TMEM24, but not other ER/PM tethers, concentrate as cell–cell junctions. HEK293 cells. (A and B) TMEM24 expressed in one cell generates enlarged ER/PM junctions that are not mirrored by eGFP-ESyt2-positive or eGFP-JPH4-positive junctions in directly adjacent cells. (C) The accumulation of TMEM24-mCherry expressed in one cell at a cell-adjacent ER/PM junction is mirrored by the accumulation of TMEM24-GFP expressed in the adjacent cell (see also ). (D) ER/PM junctions induced by mCherry-ESyt2 and GFP-ESyt2-expressed in two adjacent cells respectively, do not mirror one another across the cell–cell interface. (E) mCherry-JPH4 and GFP-JPH4 do not robustly mirror one another across the cell–cell interface although junctions could be found that seemed to be symmetrically opposed. (F) eGFP-Esyt2 colocalizes with TMEM24 at ER/PM junctions, but is excluded from the cell adjacent junction where TMEM24-GFP is selectively enriched. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (G) Kv2.1(S601,607D)-GFP, a Kv2.1 construct that binds constitutively to the ER protien VAP colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right. (H) STIM1(D76A)-GFP, a STIM1 construct that constitutively binds the PM, colocalizes with TMEM24-mCherry at all ER/PM junctions. Line scans of the fluorescence of the two constructs along the plasma membrane are shown at right.

    Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

    Techniques: Fluorescence, Construct, Membrane

    Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    doi: 10.1083/jcb.202311137

    Figure Lengend Snippet: Streptavidin affinity-purification and localization of the identified band 4.1 proteins. Anti-GFP western blots and streptavidin overlay of material affinity-purified on streptavidin bead. Numbers in parenthesis indicate amino acid boundaries of TMEM24 fragments used. Note the high degree of self-biotinylation for each construct. Source data are available for this figure: .

    Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

    Techniques: Affinity Purification, Western Blot, Construct

    A TMEM24-4.1-SynCAM1 complex at sites of cell – cell contact. (A) Schematic of the SynCAM 1(363)-GFP construct. (B) Confocal images of SynCAM1(363)-eGFP expressed in HEK293 cells. The upper panel is a slice at mid z level while the lower panel is shown as a basal surface. Representative example of n = 22 cells. (C) Coexpression of TMEM24-mCherry and SynCAM1(363)-eGFP in HEK293 cells. Images are of a mid-level z-slice (above) or a basal surface (below) of the cell. Plasma membrane labeled with CellBrite 650. Representative example of n = 35 cells. (D) . Coexpression of JPH4-mCherry and SynCAM1(363)-eGFP in HEK293 cells. The plasma membrane was labeled with CellBrite 650. Representative example of n = 28 cells (E) Pearson’s correlation R values of comparisons of the SynCAM1(363)-eGFP fluorescence with the fluorescence of either TMEM24-mCherry or mCherry-JPH4 (P = 6.76E-11, TMEM24 n = 24, JPH4 n = 30). (F) Expression of SynCAM1(363)-eGFP, mCherry-4.1G, and TMEM24-Halo labeled with JF646 HaloTag ligand in HEK293 cells. All three proteins colocalize at ER/PM junctions. Representative example of n = 74 cells. ( G ) Model of the TMEM24-4.1-SynCAM 1 complex. Diagonal lines indicate regions of the micrographs occupied by cells. Scale bars = 5 μm.

    Journal: The Journal of Cell Biology

    Article Title: A complex of the lipid transport ER proteins TMEM24 and C2CD2 with band 4.1 at cell–cell contacts

    doi: 10.1083/jcb.202311137

    Figure Lengend Snippet: A TMEM24-4.1-SynCAM1 complex at sites of cell – cell contact. (A) Schematic of the SynCAM 1(363)-GFP construct. (B) Confocal images of SynCAM1(363)-eGFP expressed in HEK293 cells. The upper panel is a slice at mid z level while the lower panel is shown as a basal surface. Representative example of n = 22 cells. (C) Coexpression of TMEM24-mCherry and SynCAM1(363)-eGFP in HEK293 cells. Images are of a mid-level z-slice (above) or a basal surface (below) of the cell. Plasma membrane labeled with CellBrite 650. Representative example of n = 35 cells. (D) . Coexpression of JPH4-mCherry and SynCAM1(363)-eGFP in HEK293 cells. The plasma membrane was labeled with CellBrite 650. Representative example of n = 28 cells (E) Pearson’s correlation R values of comparisons of the SynCAM1(363)-eGFP fluorescence with the fluorescence of either TMEM24-mCherry or mCherry-JPH4 (P = 6.76E-11, TMEM24 n = 24, JPH4 n = 30). (F) Expression of SynCAM1(363)-eGFP, mCherry-4.1G, and TMEM24-Halo labeled with JF646 HaloTag ligand in HEK293 cells. All three proteins colocalize at ER/PM junctions. Representative example of n = 74 cells. ( G ) Model of the TMEM24-4.1-SynCAM 1 complex. Diagonal lines indicate regions of the micrographs occupied by cells. Scale bars = 5 μm.

    Article Snippet: Samples were subjected to gel electrophoresis and membranes were probed with rabbit anti-GFP (cat. Ab290; Sigma-Aldrich) and mouse anti-mCherry 1C51 (cat. Ab125096; Abcam) primary antibodies overnight at 4°C.

    Techniques: Construct, Membrane, Labeling, Fluorescence, Expressing

    Intracellular AIMP2 accumulation leads to AIMP2 secretion and intercellular transmission. ( A ) Representative immunofluorescence images of endothelial marker CD31 and AIMP2 in the cortical brain subregions from two- and six-month-old AIMP2 transgenic mice and age-matched littermate controls. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar = 10 μm. ( B ) Quantification of AIMP2 signal intensities in CD31-positive brain endothelium in the indicated mouse groups ( n = 5 in two-month-age group, n = 5 in control six-month-age group, and n = 8 in AIMP2 Tg six-month-age group). ( C ) Anti-AIMP2 dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( D ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with GFP or GFP-AIMP2 based on the dot blot result in the panel C ( n = 3 separate experiments per group). ( E ) Anti-GFP dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( F ) Quantification of relative anti-GFP dot blot optical densities for experimental groups in the panel E ( n = 3 separate experiments per group). ( G ) Dot blot assessment of AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with GFP-AIMP2 construct (0, 1, 2 μg). Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( H ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with the indicated combination of GFP and GFP-AIMP2 ( n = 3 separate experiments per group). ( I ) Representative immunofluorescence images showing GFP-AIMP2 uptake into HUVECs. HUVECs were treated with conditioned media (48 h) from GFP or GFP-AIMP2 transfected SH-SY5Y cells. Scale bar = 50 μm. ( J ) Percentage GFP-positive HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). ( K ) Quantification of GFP-AIMP2 immunofluorescence signals in HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). Quantitative data are expressed as the mean ± SEM, and statistical significance was determined by ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. ns, non-significant

    Journal: Journal of Translational Medicine

    Article Title: AIMP2 accumulation in brain leads to cognitive deficits and blood secretion in Parkinson’s disease

    doi: 10.1186/s12967-024-05666-x

    Figure Lengend Snippet: Intracellular AIMP2 accumulation leads to AIMP2 secretion and intercellular transmission. ( A ) Representative immunofluorescence images of endothelial marker CD31 and AIMP2 in the cortical brain subregions from two- and six-month-old AIMP2 transgenic mice and age-matched littermate controls. The nucleus was counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar = 10 μm. ( B ) Quantification of AIMP2 signal intensities in CD31-positive brain endothelium in the indicated mouse groups ( n = 5 in two-month-age group, n = 5 in control six-month-age group, and n = 8 in AIMP2 Tg six-month-age group). ( C ) Anti-AIMP2 dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( D ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with GFP or GFP-AIMP2 based on the dot blot result in the panel C ( n = 3 separate experiments per group). ( E ) Anti-GFP dot blot assessment of GFP-AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with either GFP or GFP-AIMP2 constructs. Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( F ) Quantification of relative anti-GFP dot blot optical densities for experimental groups in the panel E ( n = 3 separate experiments per group). ( G ) Dot blot assessment of AIMP2 protein in the culture media from SH-SY5Y cells transiently transfected with GFP-AIMP2 construct (0, 1, 2 μg). Ponceau staining was used to visualize the proteins in the culture media. Complete media was changed to serum-deprived media 24 h before analysis of AIMP2 secretion. ( H ) Quantification of secreted AIMP2 in the media from SH-SY5Y cells transfected with the indicated combination of GFP and GFP-AIMP2 ( n = 3 separate experiments per group). ( I ) Representative immunofluorescence images showing GFP-AIMP2 uptake into HUVECs. HUVECs were treated with conditioned media (48 h) from GFP or GFP-AIMP2 transfected SH-SY5Y cells. Scale bar = 50 μm. ( J ) Percentage GFP-positive HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). ( K ) Quantification of GFP-AIMP2 immunofluorescence signals in HUVECs in the indicated experimental groups ( n = 3 separate experiments per group). Quantitative data are expressed as the mean ± SEM, and statistical significance was determined by ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01 and *** p < 0.001. ns, non-significant

    Article Snippet: The following primary antibodies were used: rabbit antibody against AIMP2 (Proteintech; #10424-1-AP, 1:5,000), mouse antibody against MAP2 (Sigma-Aldrich; #M4403, 1:5,000), rabbit antibody against MAP2 (Abcam; #ab32454, 1:5,000), mouse antibody against CD31 (BD Biosciences; #550274, 1:1,000), and rabbit antibody against GFP (Cell Signaling Technology; #2555S, 1:5,000).

    Techniques: Transmission Assay, Immunofluorescence, Marker, Transgenic Assay, Control, Dot Blot, Transfection, Construct, Staining

    REEP5 interacts with MFN1/2 through C-terminal cytosolic domains. All co-immunoprecipitation (Co-IP) experiments were done in HEK293T cells. (A and B) Co-IP analysis of Myc-tagged REEP 1–6 with Flag-MFN1/2. (C) Co-IP analysis of endogenous MFN1/2 with REEPs and Rtns. (D) Co-IP analysis of Flag-MFN2 wild type or Flag-MFN2 ActA with REEP5-Myc. (E) Schematic diagram of human REEP5 as previously reported ( ; ), MFN1 according to UniProt, and MFN2 according to . HR, coiled-coiled heptad repeat domain; TM, transmembrane; APH, amphipathic helix; cytL, cytosolic loop; CTH, C-terminal helix. (F and G) Co-IP analysis of mEmerald-tagged REEP5 FL or truncation mutants with Flag-MFN1/2, and GFP-tagged MFN1/2 FL or truncation mutants with REEP5-Myc. FL, full length. Numbers indicate amino acids in each construct. (H) Purified His-REEP5, GST-MFN1/2 C-terminal cytosolic domains, and GST were analyzed by Coomassie staining. (I) GST pull-down of GST-MFN1/2 C-terminal cytosolic domains with His-REEP5. Source data are available for this figure: .

    Journal: The Journal of Cell Biology

    Article Title: Dynamic interaction of REEP5–MFN1/2 enables mitochondrial hitchhiking on tubular ER

    doi: 10.1083/jcb.202304031

    Figure Lengend Snippet: REEP5 interacts with MFN1/2 through C-terminal cytosolic domains. All co-immunoprecipitation (Co-IP) experiments were done in HEK293T cells. (A and B) Co-IP analysis of Myc-tagged REEP 1–6 with Flag-MFN1/2. (C) Co-IP analysis of endogenous MFN1/2 with REEPs and Rtns. (D) Co-IP analysis of Flag-MFN2 wild type or Flag-MFN2 ActA with REEP5-Myc. (E) Schematic diagram of human REEP5 as previously reported ( ; ), MFN1 according to UniProt, and MFN2 according to . HR, coiled-coiled heptad repeat domain; TM, transmembrane; APH, amphipathic helix; cytL, cytosolic loop; CTH, C-terminal helix. (F and G) Co-IP analysis of mEmerald-tagged REEP5 FL or truncation mutants with Flag-MFN1/2, and GFP-tagged MFN1/2 FL or truncation mutants with REEP5-Myc. FL, full length. Numbers indicate amino acids in each construct. (H) Purified His-REEP5, GST-MFN1/2 C-terminal cytosolic domains, and GST were analyzed by Coomassie staining. (I) GST pull-down of GST-MFN1/2 C-terminal cytosolic domains with His-REEP5. Source data are available for this figure: .

    Article Snippet: The following antibodies were used: rabbit anti-Myc (2278; CST), rabbit anti-Flag (PM020; MBL), rabbit anti-GFP (598; MBL), rabbit anti-MFN1 (14739; CST), rabbit anti-MFN2 (11925; CST), rabbit anti-lgG (30000-0-AP; Proteintech), mouse anti-GAPDH (SC-32233; Santa Cruz), mouse anti-Tom20 (SC-17764; Santa Cruz), rabbit anti-GST (1003-8; HuaBio), rabbit anti-REEP2 (A17152; ABclonal), rabbit anti-REEP3 (1-77105; Novus NBP), rabbit anti-REEP4 (26650-1-AP; Proteintech), rabbit anti-REEP5 (A10392; ABclonal), rabbit anti-REEP6 (FNab07232; FineTest), rabbit anti-Rtn2 (11168-1-AP; Proteintech), rabbit anti-Rtn3 (A302-860A-T; Bethyl Laboratories), and rabbit anti-Rtn4 (FNab07524; FineTest).

    Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Construct, Purification, Staining