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

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Cdc6 protein levels are only detected in differentiated endoreplicating megakaryoblastic cells. (A) Exponentially growing (−) and TPA-treated (+) HEL, HA1 (Dmescargot-expressing HEL-derived line), K562 and KEB (cyclin E-overexpressing K562-derived line) cells were collected, and whole protein extracts were obtained. Total protein, 30 μg, was subjected to SDS-PAGE and detected by Western blotting with anti-cdc6, -cdt1, -geminin, and -cyclin E antibodies. A portion of total protein staining of transferred gel is shown as a loading control. Asterisk marks the faster migrating cyclin E bands, which correspond to the protein overexpressed in KEB cells. (B) DNA content pattern of these cells is shown as analyzed by flow cytometry. Vertical axis, relative number of cells; horizontal axis, relative red fluorescence (FL2) in a logarithmic scale, indicating DNA content per cell. The positions of peaks representing cells with a DNA content equal to 2C, 4C, 8C, and 16C are indicated. The ability of these cells to achieve (+), or not (−), polyploid DNA content is indicated.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: Expressing, Derivative Assay, SDS Page, Western Blot, Staining, Flow Cytometry, Fluorescence

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Cdc6 stabilization is concomitant with the establishment of the first endoreplication cycle. Whole protein extracts from HEL (A) and K562 (B) cells were obtained before treatment (0 h) or at 4, 8, 15, or 24 h after differentiation induction. Total protein, 30 μg, was subjected to SDS-PAGE and detected by Western blotting with anti-cdc6, -geminin, and -cyclin E antibodies. A portion of the total protein staining of transferred gel is shown as a loading control. Flow cytometry analysis of HEL and K562 DNA content is shown. (C) An identical experiment performed with extracts obtained form HEL and K562 cells at the indicated times after TPA treatment.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: SDS Page, Western Blot, Staining, Flow Cytometry

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Nuclear cdc6 levels are maintained in endoreplicating megakaryoblastic cells. (A) Exponentially growing (−) and 48-h TPA-treated (+) HEL, HA1, K562, and KEB cells were collected. Protein from nuclear and cytoplasmic fractions was extracted. Nuclear or cytoplasmic total protein, 100 μg, was subjected to SDS-PAGE and detected by Western blotting with anti-cdc6, -cyclin A, and -cyclin E antibodies. Blots were stripped and reincubated with anti-PCNA and −I 75 Bα antibodies to assess the purity of subcellular fractions. (B) Exponentially growing (−) or TPA-treated (+) HEL cells were collected. Cdt1 and geminin expression was detected by Western blot in both nuclear (Nuc) and cytoplasmatic (Cyt) fractions. (C) Nuclear and cytoplasmic protein extracts from exponentially growing (−) or differentiated (+) K562 and KEB cells were analyzed by Western blotting with anti-cdt1 and -geminin antibodies. Portions of the total protein staining of transferred gels are shown as loading controls.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: SDS Page, Western Blot, Expressing, Staining

Journal:

Article Title: Regulation of CDC6, Geminin, and CDT1 in Human Cells that Undergo Polyploidization

doi: 10.1091/mbc.E02-04-0217

Figure Lengend Snippet: Cdc6 is stabilized in K562 cells with high levels of cyclin E. (A) KEB cells were cultured for 48 h in the absence (−) or the presence (+) of TPA and total RNA was extracted. RNA, 20 μg, was analyzed by Northern blot with full-length cdc6 cDNA as a probe. The same blot was incubated with a probe recognizing 28S ribosomic RNA and is shown as a loading control. (B) K562 and KEB cells were transiently transfected with either pMT or pMTcdc6 plasmids. Nontransfected, pMT-transfected, and pMTcdc6-transfected cells were allowed to grow exponentially (−) or treated with TPA for 48 h (+). Whole protein fractions were obtained, and Western blot analysis with anticdc6 antibody was performed. An overexposed film is also shown to stress the limited expression of myc-tagged cdc6. (C) pMTcdc6-transfected KEB cells, 2 × 104, were cytospun, fixed with formaldehyde, permeabilized with TX100, and incubated sequentially with anti-myc 9E10 and Alexa Fluor 488–conjugated anti-mouse Ig antibodies. Nuclei were counterstained with DAPI. The figure shows the maximal projection (blue, green, and merge) of an image at 1024 × 1024 resolution. (D) K562 cells were transiently cotransfected with pMTcdc6 and pcDNA-cycE or the corresponding empty plasmids. Either exponentially growing (−) or TPA-treated (+) cells were analyzed by Western blot using anti-cdc6 and -cyclin E antibodies. (E) Western blot analysis with anti-cdc6 and -cyclin E antibodies of TPA-treated pMT-, pMT-cdc6 wild-type (pMTwt)- or pMT- cdc6 5xA mutant (pMT5xA)-transfected cells, and cells cotransfected with pMTwt or pMT5xA plasmids, together with pCDNA-cyclin E are shown. Portions of the total protein staining of transferred gels are shown as loading controls. Both D and E show overexposed blots.

Article Snippet: Antibodies used were as follows: anti-human cdc6 mouse mAb (Ab3; Oncogene, Darmstadt, Germany) at a 1:500 dilution; anti-human cdt1 rabbit polyclonal antibody ( Nishitani et al. , 2000 ) at a 1:2500 dilution; anti-human geminin goat polyclonal antibody (C-16; Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution; anti-human cyclin E mouse mAb (PharMingen) at a 1:1000 dilution; and anti-human cyclin A rabbit polyclonal antibody (Santa Cruz Biotechnology) at a 1:1000 dilution.

Techniques: Cell Culture, Northern Blot, Incubation, Transfection, Western Blot, Expressing, Mutagenesis, Staining