Journal: The Journal of Cell Biology

Article Title: Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

doi: 10.1083/jcb.201601071

Figure Lengend Snippet: Core-shell arrangement of protein components in paraspeckle spheres I. (A) Simultaneous detection of Neat1 and seven of the protein components of paraspeckles, including Sfpq, Nono, Pspc1, Fus, Rbm14, Brg1, and Tardbp in corpus luteal cells. Note that the paraspeckle proteins are grouped into the core, patch, and shell components depending on their distribution in the paraspeckles. (B) Dendrogram based on pairwise class-distance matrix generated using the machine-learning pattern-recognition tool wndchrm. The shell, core, and patch components are grouped into three distinct branches. (C) A model for the structure of paraspeckles. Neat1 folds in half with the 5′ and the 3′ regions bundled independently and radially arranged to construct scaffolds of paraspeckles. Bar, 500 nm.

Article Snippet: For the simultaneous detection of paraspeckle proteins, the following antibodies were used: mouse mAb against Sfpq (clone B92; Abcam), mouse mAb against Nono , mouse mAb against Pspc1 (clone 1L4; Sigma-Aldrich), mouse mAb against Fus (clone 4H11; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody against Fus (ab84078; Abcam), rabbit polyclonal antibody against Brg1 (A300-813A; Bethyl Laboratories, Inc.), rabbit polyclonal antibody against Tardbp (10782-2-AP; Proteintech), and rabbit polyclonal antibody against Rbm14 (A300-311A; Bethyl Laboratories, Inc.).

Techniques: Generated, Construct

Journal: The Journal of Cell Biology

Article Title: Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

doi: 10.1083/jcb.201601071

Figure Lengend Snippet: Core-shell arrangement of protein components in paraspeckle spheres II. Higher magnification SIM images of two of the representative single paraspeckles stained with the Neat1 5′+3 ′ probe and Sfpq (A), Nono (B), Pspc1 (C), Fus (D), Rbm14 (E), Brg1 (F), and Tardbp (G). Intensity profiles along the dashed lines (a and b) are shown in the graphs next to the images. Bar, 100 nm.

Article Snippet: For the simultaneous detection of paraspeckle proteins, the following antibodies were used: mouse mAb against Sfpq (clone B92; Abcam), mouse mAb against Nono , mouse mAb against Pspc1 (clone 1L4; Sigma-Aldrich), mouse mAb against Fus (clone 4H11; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody against Fus (ab84078; Abcam), rabbit polyclonal antibody against Brg1 (A300-813A; Bethyl Laboratories, Inc.), rabbit polyclonal antibody against Tardbp (10782-2-AP; Proteintech), and rabbit polyclonal antibody against Rbm14 (A300-311A; Bethyl Laboratories, Inc.).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

doi: 10.1083/jcb.201601071

Figure Lengend Snippet: Fus-independent and dependent recruitment of paraspeckle proteins. (A) Simultaneous detection of Neat1 and seven of the protein components of paraspeckles, including Sfpq, Nono, Pspc1, Fus, Rbm14, Brg1, and Tardbp, in MEFs derived from WT and Fus KO mice. Note that DBHS family proteins (Sfpq, Nono, and Pspc1) and Tardbp, but not Rbm14 and Brg1, are recruited to the putative transcription site in the absence of Fus. Arrowheads indicate paraspeckle-like nuclear bodies formed at the putative Neat1 transcription site in Fus KO MEFs. (B) Simultaneous detection of various forms of NEAT1 and NONO in HAP1 cells and FUS-deleted HAP1 cells (ΔFUS HAP1). Probes used to detect NEAT1 are shown in the top boxes. (C) Schematic drawing of full-length and mutant FUS protein exogenously expressed by lentiviruses. ΔN FUS lack the PrLD and ΔC FUS lack the RNA binding domains including RNA recognition motifs (RRM) and arginine (R)-glycine-glycine domain (RGG) as well as zinc finger domain (ZF). (D) Western blot analyses of lysate from the cells infected with control EGFP (C), full-length FUS (FL), ΔN FUS (ΔN), and ΔC FUS (ΔC). Note that migration of FL and ΔN are much slower than predicted molecular mass (57 and 35 kD, respectively), probably because of the presence of PrLD in these molecules. (E) Simultaneous detection of Neat1 5′+3′ and Nono in Fus KO MEFs expressing various forms of FUS protein. Note that the core-shell structure of paraspeckles was rescued with FL FUS, but not with mutant molecules that lack either PrLD or RNA binding domains. (F) Confirmation of the specificity of polyclonal [Fus (poly)] and monoclonal (Fus) antibodies against Fus. Mixtures of MEFs derived from WT and KO mice of Fus were stained with each antibody. Note the complete absence of signals in the Fus KO MEFs (arrowheads). The positions of the epitope of these antibodies are shown in the schematic drawing of the domain structure of Fus. (G) Simultaneous detection of Fus using polyclonal antibodies and mAbs that recognize the N- and C-terminal region of the protein, respectively. Bars: (A, B, E, and G) 500 nm; (F) 200 µm.

Article Snippet: For the simultaneous detection of paraspeckle proteins, the following antibodies were used: mouse mAb against Sfpq (clone B92; Abcam), mouse mAb against Nono , mouse mAb against Pspc1 (clone 1L4; Sigma-Aldrich), mouse mAb against Fus (clone 4H11; Santa Cruz Biotechnology, Inc.), rabbit polyclonal antibody against Fus (ab84078; Abcam), rabbit polyclonal antibody against Brg1 (A300-813A; Bethyl Laboratories, Inc.), rabbit polyclonal antibody against Tardbp (10782-2-AP; Proteintech), and rabbit polyclonal antibody against Rbm14 (A300-311A; Bethyl Laboratories, Inc.).

Techniques: Derivative Assay, Mutagenesis, RNA Binding Assay, Western Blot, Infection, Control, Migration, Expressing, Staining

Journal: Acta Neuropathologica Communications

Article Title: Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

doi: 10.1186/s40478-019-0865-5

Figure Lengend Snippet: Organotypic mouse hippocampal slice cultures as a model to study seeded α-syn aggregation in the region between DG and CA3. a Diagram showing the synaptic connections of granule cells of DG (where S129A PFFs were injected) to pyramidal neurons in CA3 that subsequently connect to the pyramidal neurons of the CA1 region. b OHSCs from mouse pups were cultivated on an air-liquid interface. c Progressive accumulation of total ( i ) and pS129-α-syn ( ii , 11A5) in cultures from wild type mouse pups after 0, 7, 14, and 21 DIV analyzed by immunoblotting. d Experimental flow showing time of PFF microinjection at 7 DIV and tissue collection for analysis at 3, 5, 7, and 14 dpi. e pS129-positive α-syn structures (D1R1R) imaged at DG, following PFF injection at DG. Aggregates are first recognizable at 3 dpi as short serpentine aggregates ( i ) that coalesce into longer aggregates by 5 dpi ( ii ) and at 7 dpi occur as fibrillar aggregates around neuronal nuclei ( iii ). Scale bars: 20 μm. f MJF-14-positive serpentine aggregates co-localize with the axonal marker neurofilament light chain (NF-L). Scale bar: 20 μm. g pS129-positive cell body inclusions (D1R1R) are located in NeuN-positive neurons. Scale bar: 20 μm, inset: 5 μm. h Thread-like cell body inclusion detected by MJF-14 and reconstructed in 3D by IMARIS software. i Cell body pS129 α-syn pathology (D1R1R) in the hindbrain of end-stage h-A53T-α-syn transgenic mice (M83) resembles inclusions in the slice model (panels e,iii & g ). j dSTORM image reconstruction of pS129-positive axonal processes (D1R1R) within the OHSC. Scale bar: 1 μm. k Progressive accumulation of insoluble pS129-positive mouse α-syn (11A5) in PFF-injected slices. Western blots in c & k are representative of 2–3 separate experiments. Images in e are examples from 2 to 6 individual experiments with 9–17 slices in total. Images in f & g are representative of 4–5 experiments/15–16 slices in total

Article Snippet: Antibodies used were the following: rabbit polyclonal anti-α-syn (ASY-1 1:1000) [ ], rabbit mAb anti-α-syn antibody (MJFR1 #ab138501, Abcam, 1:1000), mouse mAb pS129-α-syn (11A5, kindly provided by Imago Pharmaceuticals, 1:2000), mouse mAb anti-β-Tubulin III (TUJ1 #T8578, Sigma, 1:5000), rabbit mAb mouse-specific α-syn (D37A6 XP Rabbit #4179, Cell Signaling, 1:1000), mouse mAb anti-α-syn Syn-1 (Clone 42 #610787, BD Transduction Laboratories, 1:1000).

Techniques: Injection, Western Blot, Marker, Software, Transgenic Assay

Journal: Acta Neuropathologica Communications

Article Title: Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

doi: 10.1186/s40478-019-0865-5

Figure Lengend Snippet: Trans-synaptic spreading of α-syn aggregate pathology from DG via CA3 to the CA1 region depends on α-syn expression levels. a No aggregation is induced by injection of ( i ) monomeric α-syn in WT slices or ( ii ) S129A PFFs in α-syn KO slices. Scale bars: 20 μm. b Composite image of immunostaining for aggregated (MJF-14, green) and pS129-α-syn (11A5, red) 7 dpi in WT OHSCs, scale bar: 200 μm. Areas from DG, CA3, and CA1 regions indicated are magnified in panels i , ii , and iii . Scale bars: 20 μm. Axonal aggregates (arrows) are present in all three regions, while cell body inclusions (arrowheads) are present only in DG at 7 dpi. c Composite image of immunostaining with MJF-14 and pS129 for aggregates 7 dpi in ASO OHSCs. Scale bar: 200 μm. i , ii Extensive MJF-14- and pS129-positive aggregation and ( iii ) faster progression with development of cell body inclusions in the CA1 region. Scale bars: 20 μm. d Quantification of pS129-α-syn aggregate fluorescence signals in total slices from PFF-injected WT and ASO slices. Bars represent mean ± SD, n = 3. Unpaired Student’s T-test, p -value = 0.019. e Immunostaining with pS129 (11A5) and MJF-14 at CA1 region of WT slices 14 dpi of PFFs show more compacted, spherical cytoplasmic inclusions, resembling Lewy bodies. Scale bar: 5 μm. f Schematic presentation of progressive development of aggregation; from short into longer serpentine, axonal inclusions in DG regions, which spread to CA3 and CA1 regions. Cell body inclusions appear at later stages when axonal pathology is already established in the region. Images in a are illustrative of 2–3 individual experiments with 10–12 slices in total. Images in b are representative of 17 slices/6 experiments, while images in c represent 3 slices/1 experiment. For quantification in d , 3 slices were included per group

Article Snippet: Antibodies used were the following: rabbit polyclonal anti-α-syn (ASY-1 1:1000) [ ], rabbit mAb anti-α-syn antibody (MJFR1 #ab138501, Abcam, 1:1000), mouse mAb pS129-α-syn (11A5, kindly provided by Imago Pharmaceuticals, 1:2000), mouse mAb anti-β-Tubulin III (TUJ1 #T8578, Sigma, 1:5000), rabbit mAb mouse-specific α-syn (D37A6 XP Rabbit #4179, Cell Signaling, 1:1000), mouse mAb anti-α-syn Syn-1 (Clone 42 #610787, BD Transduction Laboratories, 1:1000).

Techniques: Expressing, Injection, Immunostaining, Fluorescence

Journal: Acta Neuropathologica Communications

Article Title: Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

doi: 10.1186/s40478-019-0865-5

Figure Lengend Snippet: Application I. Demonstrating trans-synaptic spreading as a route for spreading of α-syn-aggregate pathology from DG via CA3 to the CA1 region using surgical and viral transgene methods. a Illustration of PFF injection in CA1 in WT OHSCs to test the efficiency of the retrograde route of spreading. 1 Composite image 14 dpi of S129A PFFs at CA1. Scale bar: 200 μm. MJF-14-positive aggregates are seen at the CA1 region ( i, ii ), but there is no spreading to DG ( iii) . Scale bar i & iii: 50 μm, ii: 20 μm. b Diagram showing transection of axonal projections between DG and CA3, which blocks spreading of α-syn aggregate pathology from DG to CA1. The surgical destruction of the tissue is demonstrated by the absence of nuclei ( 1 ), axonal marker NF-L ( 2 ), and MJF-14 staining ( 3 ). Scale bars: 200 μm. Magnified images from 3 show aggregates at DG ( i ) and proximal to the cut ( ii ), but not distal to the lesion ( ii, iii ). Scale bars: 50 μm. c Diagram showing expression of WT - α-syn in α-syn KO slices by AAV vectors injected in DG, CA3, and CA1. 1 α-syn expression in DG, CA3, and CA1 supports spreading of aggregated pS129 α-syn (11A5) to CA1 7 dpi of PFFs in DG, as seen from the magnified panels i - iiii . Scale bar: 200 μm, i & iiii: 20 μm, ii & iii: 10 μm. Note the strong AAV-dependent expression of pS129 in some neuronal nuclei. d Illustration of WT - α-syn expression in DG and CA1 only of α-syn KO slices. 1 Absence of α-syn expression in the CA3 abolishes spreading of aggregation to CA1 at 7 dpi. Scale bar: 200 μm. i, ii pS129-positive aggregates are detectable at DG. iii N o pS129-positive aggregates are found at the CA1 region. Iiii A few neurons show nuclear expression of pS129-α-syn at CA3. Scale bars: i: 20 μm, ii, iii & iiii: 10 μm. Data in a are illustrative of 12 slices divided over 3 experiments. Images in b are representative of 4 experiments with 18 slices in total, while c & d representative of 3 separate experiments/18–21 slices in total per condition

Article Snippet: Antibodies used were the following: rabbit polyclonal anti-α-syn (ASY-1 1:1000) [ ], rabbit mAb anti-α-syn antibody (MJFR1 #ab138501, Abcam, 1:1000), mouse mAb pS129-α-syn (11A5, kindly provided by Imago Pharmaceuticals, 1:2000), mouse mAb anti-β-Tubulin III (TUJ1 #T8578, Sigma, 1:5000), rabbit mAb mouse-specific α-syn (D37A6 XP Rabbit #4179, Cell Signaling, 1:1000), mouse mAb anti-α-syn Syn-1 (Clone 42 #610787, BD Transduction Laboratories, 1:1000).

Techniques: Injection, Marker, Staining, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

doi: 10.1186/s40478-019-0865-5

Figure Lengend Snippet: Application II. Demonstrating that phosphorylation of S129 on α-syn is not a prerequisite for seeding α-syn aggregation or trans-synaptic spreading in hippocampal slices. a Experimental setup with establishment of neuronal expression of either WT- or non-phosphorylatable S129G-α-syn in α-syn KO slices prior to initiation of templated α-syn aggregation by injection of S129A PFFs. b Validation of virally mediated WT- and non-phosphorylatable S129G-α-syn expression in α-syn KO slices using antibodies against total and pS129-α-syn (11A5). c Expression of WT α-syn supports establishment of MJF-14- and pS129-positive (11A5) aggregate pathology in the DG region following PFF injection at DG. Magnified panels show axonal aggregates ( i ) and cell body inclusions ( ii ) at DG. Scale bar: 50 μm, i : 20 μm, ii : 5 μm. d MJF-14- and pS129 positive (11A5) pathology spreads to the CA1 region within 7 dpi. Scale bar: 50 μm. e Expression of S129G-α-syn supports establishment of MJF-14-positive/pS129-negative aggregate pathology in the DG, present in axons ( i , arrows) and cell bodies ( i , arrowheads). Scale bar: 50 μm, i : 20 μm. f The non-phosphorylated MJF-14-positive aggregate pathology spreads to the CA1 region within 7 dpi. Scale bar: 50 μm. Western blot in b is representative of 3 independent experiments, while images in c - f are illustrative of 3–5 experiments with 21–30 slices in total per condition

Article Snippet: Antibodies used were the following: rabbit polyclonal anti-α-syn (ASY-1 1:1000) [ ], rabbit mAb anti-α-syn antibody (MJFR1 #ab138501, Abcam, 1:1000), mouse mAb pS129-α-syn (11A5, kindly provided by Imago Pharmaceuticals, 1:2000), mouse mAb anti-β-Tubulin III (TUJ1 #T8578, Sigma, 1:5000), rabbit mAb mouse-specific α-syn (D37A6 XP Rabbit #4179, Cell Signaling, 1:1000), mouse mAb anti-α-syn Syn-1 (Clone 42 #610787, BD Transduction Laboratories, 1:1000).

Techniques: Expressing, Injection, Western Blot

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) C33A cells expressing EV, HCD13 or Y6F were treated with vehicle (Veh) or aluminum fluoride (AlF; 50 μM) for 30 min, and membrane fractions were purified from total cell lysates. Active ARF6-GTP in the fractions was measured by pull down assay and normalized to total ARF6. Validation of the membrane fraction was verified by expression of plasma membrane marker E-cadherin. (B) Plots depict quantification of immunoblot analysis of ARF6-GTP, CD13, and IQGAP1 normalized to E-cadherin in the plasma membrane. Data are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 by two-tailed student’s t test.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Expressing, Purification, Pull Down Assay, Marker, Western Blot, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A) Lysates from C33A cells expressing EV, HCD13 or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1 and total ARF6 by immunoblot analysis. ARF6-GTP was detected by a pull-down assay of the immunoprecipitates using beads conjugated to the PBD binding domain of GGA3 for 1 hour at 4°C. (B) Lysates from T27N ARF6 dominant-negative mutant cells expressing EV, HCD13 or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1, ARF6 or HA-tag by immunoblot analysis. Active ARF6 was detected by pull down with GGA3-conjugated beads from the immunoprecipitates. (C and D) ARF6 activity was measured in C33A cells expressing EV or HCDi3 after cell-ECM adhesion over the indicated time. Using ARF6 protein-binding domain (PBD) of the effector protein GGA3 conjugated beads, which specifically binds the GTP-bound form of ARF6, the subsequent pull-down of ARF6-GTP was quantified by immunoblot analysis using the ARF6-specific antibody. Blots (C) are representative and quantified data (D) are means ± SD of 3 independent experiments. (E and F) β1-integrin receptor recycling was measured in a pulse-chase assay with β1 integrin Ab clone 12G10 (for C33A cells; E) or 9EG7 (for MEFs; F) in the presence of N-myristolated ARF6 inhibitor peptide. Serum-starved cells were treated with β1-integrin Ab at 4°C for 30 min, pulsed for 1 hour to induce internalization, acid-stripped, washed, treated with myr-ARF6 peptide (10 μM) for 30 min, and allowed to recycle at 37°C for 2 to 4 hours. Paraformaldehyde-fixed cells were stained with fluorescently conjugated secondary Ab and MFI (Mean Fluorescence Intensity) of surface β1-integrin was analyzed by flow cytometry. Data are mean ± SD of 3 independent experiments. *P<0.05 and **P<0.0i by two-tailed student’s t test.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Binding Assay, Dominant Negative Mutation, Activity Assay, Protein Binding, Pulse Chase, Staining, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: (A and B) In a scratch assay, following injury on the monolayer by creating a scratch, C33A cells expressing HCD13 were allowed to migrate to the wound and fixed with 4% paraformaldehyde at the 6-hour time point. Cells were stained with phalloidin (red; left rows) or CD13 (red; right rows) and IQGAP1 (green) and imaged using confocal microcopy; magnified inset of CD13/IQGAP1-stained C33A-HCD13 cells is shown (B). Scale bar; 5μm. DAPI (blue). (C) Quantification of the area of F-actin and IQGAP1 accumulation at the migrating front of the cell, represented in (A), normalized to total cell area by Fiji software. Five fields were counted for each genotype, and all cells in each field were measured. (D) Percent of F-actin+, CD13+ and IQGAP1+ cells from (A) at the leading front were measured in each of five fields for each genotype. Data are mean ± SD of 3 independent experiments. *P<0.05 by two-tailed student’s t test.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Wound Healing Assay, Expressing, Staining, Software, Two Tailed Test

Journal: Science signaling

Article Title: CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration

doi: 10.1126/scisignal.aav5938

Figure Lengend Snippet: In wild-type cells, phospho-CD13 and β1-integrin internalize into early endosomes, sort to recycling endosomes and return to the cell membrane, enabling cell-ECM adhesion and migration. However, in cells lacking CD13 or expressing an inactive CD13 mutant, whereas β1-integrin internalizes into early endosomes, it aberrantly traffics to Rab7+ lysosomes and it is ultimately degraded. Mechanistically, CD13 must be present in a complex containing the scaffolding protein IQGAP1, active-ARF6 its GEF EFA6 and β1-integrin at the plasma membrane to allow proper β1-integrin recycling and cell migration to proceed. In the absence of CD13, no active-ARF6 is detected in the plasma membrane and IQGAP1 is not recruited to the migrating front, thereby diminishing cell adhesion, spreading and migration.

Article Snippet: Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Techniques: Migration, Expressing, Mutagenesis, Scaffolding

Journal: bioRxiv

Article Title: Family-wide analysis of integrin structures predicted by AlphaFold2

doi: 10.1101/2023.05.02.539023

Figure Lengend Snippet: ( A ) Structures of α 10 integrins from human, mouse, rat, and zebrafish. The structures were aligned based on the calf-2 domain and oriented perpendicularly to the cell membrane. The N-glycan site at the thigh/calf-1 interface is shown as a red stick. ( B ) Relative positions of α 10 thigh and calf-1 domains in the fully bent conformation, illustrating the interfacial location of the N-glycan site shown as red sticks. The thigh and calf-1 domains of α 10 were superimposed on those of bent α IIb integrin structure. ( C ) Structures of β 1 integrins from human, mouse, cat, and chicken. The structures were aligned based on the βI domain and oriented perpendicularly to the cell membrane. ( D ) Conformation of β 1 integrin co-expressed with selected integrin α subunit. Human integrin α subunits with a C-terminal EGFP tag were co-expressed with human β 1 in 293T cells. The binding of mAb 9EG7 or MAR4 was measured by flow cytometry in a buffer containing 1 mM Ca 2+ /Mg 2+ or 0.1 mM Ca 2+ plus 2 mM Mn 2+ . The data are presented as the MFI of 9EG7 binding as a percentage of the MFI of MAR4 binding.

Article Snippet: The mouse mAb MAR4 (cat# 555442, BD Biosciences) was used to measure total surface expression of β 1 integrin.

Techniques: Membrane, Binding Assay, Flow Cytometry

Journal:

Article Title: Expression artifact with retroviral vectors based on pBMN

doi: 10.1016/j.ab.2009.07.014

Figure Lengend Snippet: A. The structures of the p120-1A and p120-1AC constructs are shown. In addition to p120-1A, p120-1AC contains extra 6 amino acids encoded by exon C in the middle of the Armadillo repeats. The N-terminal HA epitope and the epitope recognized by pp120 are shown.

Article Snippet: Anti-p120 mouse mAb (pp120) was purchased from BD Biosciences (Franklin Lakes, NJ).

Techniques: Construct