Journal: Development (Cambridge, England)

Article Title: Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1.

doi: 10.1242/dev.024133

Figure Lengend Snippet: Fig. 2. Netrin 1 inhibits RhoA in SCNs. G-LISA (A) and GST-RBD pulldown (B) assays were used to evaluate the relative amounts of RhoA-GTP in SCNs. GST-RBD pulldown assays measured a 27% (P=0.040) average reduction across three separate experiments. G-LISA assays measured an average reduction of 13% after 15 minutes of 200 ng/ml netrin 1 (n=23, P=0.002). This was blocked with either 5 μg/ml of DCC function blocking antibodies (DCC-fb) or 2 μg/ml of the extracellular domain of DCC (DCC-fc). Tukey post-hoc tests of means. Error bars indicate s.e.m.

Article Snippet: *Author for correspondence (e-mail: timothy.kennedy@mcgill.ca) Accepted 28 June 2008 D E V E LO P M E N T 2856 Tag1 (TG3) for western blot analysis (provided by Dr Thomas Jessell, Columbia University, New York, NY); rabbit anti-integrin β1 (AB1952, Chemicon, Temecula, CA); mouse IgG anti-RhoA (26C4) and goat antiDCC (A-20, Santa Cruz Biotechnology, Santa Cruz, CA); mouse IgG antiDCC (AF5) and Y27632 (Calbiochem, LaJolla, CA); DCC-fc, a protein chimera composed of the extracellular domain of mouse DCC and the Fc portion of human IgG1 (R & D Systems, Minneapolis, MN); mouse IgG anti-ROCKII (BD Biosciences, Mississauga, Canada); rabbit anti-PRK2 (Cell Signaling, Danvers, MA); DNase, poly-D-lysine (PDL, 70-150 kDa) and Hoechst 33258 (Sigma-Aldrich, Mississauga, Canada); and Neurobasal, FBS, B-27 supplement, GlutaMAX-1, penicillin-streptomycin, Ca2+/Mg2+free HBSS, Alexa 546-coupled phalloidin and Jasplakinolide were purchased from Invitrogen Canada (Invitrogen Canada, Burlington, ON).

Techniques: Blocking Assay

Journal: Development (Cambridge, England)

Article Title: Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1.

doi: 10.1242/dev.024133

Figure Lengend Snippet: Fig. 7. Inhibiting RhoA signaling promotes adhesion and growth cone expansion in response to netrin 1. (A) SCN adherence to netrin 1 is increased by 79% (n=12, P<0.001) in the presence of C3-07 (C3) or by 152% (n=12, P<0.001) with Y-27632 (Y). This adhesion was reduced (n=12, P<0.001) upon pre-incubation with either: netrin function blocking antibodies (anti-netrin) or by competition with the extracellular domain of DCC (DCC-fc receptor-body). Stabilization of filamentous actin with jasplakinolide (Jasp.) disrupted adhesion to netrin 1 and abolished the increased adhesion evoked by C3-07 or Y-27632. (B-D) Representative images of cells binding to netrin 1 substrates in the absence (B) or in the presence of C3-07 (C) or Y-27632 (D). (E-H) The expansion of SCN growth cones as they encounter a boundary of immobilized netrin 1 is consistent with netrin 1 functioning as an adhesive cue. (I-N) SCNs labeled with phalloidin (green), β-tubulin (red) and Hoechst (blue). The growth cones of SCNs grown on glass coverslips coated with PDL (I-K) are smaller than those grown on this same substrate with an additional coating of netrin 1 (L-N). (O) In the absence of netrin 1, the average growth cone increased by 67% (n=21, P=0.008) when treated with C3-07 and by 79% (n=20, P<0.001) when treated with Y27632. On a substrate of netrin 1, average growth cone area increased by 94% in the absence of inhibitors. A netrin 1 substrate also increased the average area of growth cones in the presence of C3-07 by 76% (n=22, P=0.006) and Y27632 by 70% (n=21, P=0.033). SCN were infected with herpes simplex viral vectors encoding with RFP (P), wt-RhoA (Q) or ca-RhoA (R). Neurons were visualized with Hoechst (blue), phalloidin (green) and with antibodies against myc (red) or endogenous RFP fluorescence (red). Growth cones area (S) was reduced by 61% when RhoA was overexpressed (n=37, P<0.001) or by 90% upon expression of ca-RhoA (n=20, P<0.001). (T) Working model illustrating the hypothesis that RhoA inhibition by netrin 1 enhances chemoattraction by facilitating DCC function, in part by recruiting additional DCC to the plasma membrane and by promoting DCC signaling mechanisms, such as increasing adhesion to immobilized netrin 1, that lead to membrane extension. Tukey post-hoc tests of means. Error bars indicate s.e.m. Scale bars: 200 μm in B-D; 20 μm in E-H; 10 μm in I-R. (A,O,S) *P<0.05; **P<0.01. (O) For the fifth and eighth bars, **P<0.01 relative to the second bar. For the third, sixth and ninth bars, *P<0.05 and **P<0.01 relative to the second, fifth and eighth bars, respectively (i.e. compared with the absence of DCC-fc).

Article Snippet: *Author for correspondence (e-mail: timothy.kennedy@mcgill.ca) Accepted 28 June 2008 D E V E LO P M E N T 2856 Tag1 (TG3) for western blot analysis (provided by Dr Thomas Jessell, Columbia University, New York, NY); rabbit anti-integrin β1 (AB1952, Chemicon, Temecula, CA); mouse IgG anti-RhoA (26C4) and goat antiDCC (A-20, Santa Cruz Biotechnology, Santa Cruz, CA); mouse IgG antiDCC (AF5) and Y27632 (Calbiochem, LaJolla, CA); DCC-fc, a protein chimera composed of the extracellular domain of mouse DCC and the Fc portion of human IgG1 (R & D Systems, Minneapolis, MN); mouse IgG anti-ROCKII (BD Biosciences, Mississauga, Canada); rabbit anti-PRK2 (Cell Signaling, Danvers, MA); DNase, poly-D-lysine (PDL, 70-150 kDa) and Hoechst 33258 (Sigma-Aldrich, Mississauga, Canada); and Neurobasal, FBS, B-27 supplement, GlutaMAX-1, penicillin-streptomycin, Ca2+/Mg2+free HBSS, Alexa 546-coupled phalloidin and Jasplakinolide were purchased from Invitrogen Canada (Invitrogen Canada, Burlington, ON).

Techniques: Incubation, Blocking Assay, Binding Assay, Adhesive, Labeling, Infection, Fluorescence, Expressing, Inhibition, Clinical Proteomics, Membrane

Journal: Open Biology

Article Title: ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis

doi: 10.1098/rsob.150218

Figure Lengend Snippet: ESCRT-II regulates surface levels of DCC in growth cones. ( a–d ) Immunostaining for total ( a,c ) and surface ( b,d ) DCC receptor in control ( a,b ) and ESCRT-II-depleted ( c,d ) GCs. For clarity, the signal intensities in ( b,d ) are increased by 30% compared with ( a,c ). GCs are outlined with yellow lines. ( e–g ) Graphs showing quantification of total ( e ) and surface ( f ) DCC levels and surface to total DCC ratios ( g ), normalized to the respective controls. * p ≤ 0.05 Mann–Whitney test. Scale bars, 10 µm.

Article Snippet: The pair of primary antibodies, either mouse anti-DCC intracellular domain (1 : 100, BD Biosciences) and rabbit anti-ESCRT-II (1 : 200) or the same anti-DCC and rabbit anti-RPL5 (1 : 100, Proteintech Europe cat. no. 15430-1-AP), were applied and incubated overnight at 4°C.

Techniques: Immunostaining, MANN-WHITNEY

Journal: Open Biology

Article Title: ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis

doi: 10.1098/rsob.150218

Figure Lengend Snippet: ESCRT-II co-localizes with DCC in growth cones. ( a–c ) Co-localization of ESCRT-II ( a ; red in c ) and DCC ( b ; green in c ) immunofluorescent signals in Xenopus RGC GCs. The signal overlap is especially visible in filopodia (indicated with arrows on insets below). ( d ) Table shows Manders' co-localization of DCC and ESCRT-II in whole GC and filopodia. ( e–g ) Proximity ligation assay confirming the close localization of ESCRT-II and DCC in RGC GCs ( e ). Yellow dots denote the sites where both probes interact. The known interaction of DCC with the large ribosomal subunit protein RPL5 was used as a positive control ( f ). GCs are outlined with yellow lines. Graph ( g ) shows the quantification of the number of PLA puncta per unit area. * p ≤ 0.05, Student's t -test. Scale bars, 10 µm.

Article Snippet: The pair of primary antibodies, either mouse anti-DCC intracellular domain (1 : 100, BD Biosciences) and rabbit anti-ESCRT-II (1 : 200) or the same anti-DCC and rabbit anti-RPL5 (1 : 100, Proteintech Europe cat. no. 15430-1-AP), were applied and incubated overnight at 4°C.

Techniques: Proximity Ligation Assay, Positive Control

Journal: Open Biology

Article Title: ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis

doi: 10.1098/rsob.150218

Figure Lengend Snippet: ESCRT-II regulates the levels of DCC receptor in growth cones. ( a–h ) ESCRT-II knockdown leads to decreased DCC levels in GCs. ( a–f ) Representative examples of GCs from embryos injected with control MO ( a,b ), ESCRT-II MO ( c,d ) and ESCRT-II MO + ESCRT-II mRNAs ( e,f ), stained for ESCRT-II ( a,c,e ) and DCC ( b,d,f ). ( g,h ) Graphs showing the normalized signal intensities of ESCRT-II ( g ; black bars) and DCC ( h ; white bars). *** p ≤ 0.0001 compared with control, Students' t -test. ( i ) A representative western blot from eye extracts indicating that the decrease in DCC levels shown in ( b,d ) is global. GCs are outlined with yellow lines. Scale bars, 10 µm.

Article Snippet: The pair of primary antibodies, either mouse anti-DCC intracellular domain (1 : 100, BD Biosciences) and rabbit anti-ESCRT-II (1 : 200) or the same anti-DCC and rabbit anti-RPL5 (1 : 100, Proteintech Europe cat. no. 15430-1-AP), were applied and incubated overnight at 4°C.

Techniques: Injection, Staining, Western Blot

Journal: Open Biology

Article Title: ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis

doi: 10.1098/rsob.150218

Figure Lengend Snippet: DCC rescues ESCRT-II knockdown phenotypes. ( a–d ) In vivo ventral view of the Xenopus optic path in stage 41 embryos whose right eye had been electroporated with control MO ( a ), ESCRT-II MO ( b ) and ESCRT-II MO + DCC mRNA ( c ). The numbers of axons exiting the eye and navigating in the optic pathway were counted and the quantification is shown in ( d ). OT, optic tract; OC, optic chiasm; tec, optic tectum. ( e–o ) In vitro turning assay. ( e–j ) Representative examples of RGC axons from embryos injected with control MO ( e,h ), ESCRT-II MO ( f,i ) and ESCRT-II MO + DCC mRNA ( g,j ) before ( e–g ) and after ( h–j ) being subjected to a Netrin-1 gradient ejected from a pipette (indicated with black arrowheads) set at 45° angle from the direction of growth. Growth measurement start point is indicated with horizontal black dotted line; dashed lines show the measured directions of growth at time 0 min and 45 min. ( k–m ) Traces of control ( k ), ESCRT-II MO ( l ) and ESCRT-II MO + DCC mRNA ( m ) axons growing for 1 h while exposed to Netrin-1 gradient (black arrowheads). ( n ) Cumulative distribution plot showing the turning angles of all measured axons. * p ≤ 0.05, ANOVA + uncorrected LSD Fisher's test. Scale bars, 20 µm.

Article Snippet: The pair of primary antibodies, either mouse anti-DCC intracellular domain (1 : 100, BD Biosciences) and rabbit anti-ESCRT-II (1 : 200) or the same anti-DCC and rabbit anti-RPL5 (1 : 100, Proteintech Europe cat. no. 15430-1-AP), were applied and incubated overnight at 4°C.

Techniques: In Vivo, In Vitro, Injection, Transferring

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Netrin-1 elicits exocytosis and axonal guidance depending on Sytx1 and TI-VAMP. A–C, Confocal images of live hippocampal growth cones traced with BODIPY (recorded in the red channel). Neurons were labeled with BODIPY ceramide for 30 min at room temperature and then chased for 2.5–3 h at 37°C. Growth cones were treated with control medium, Netrin-1, or Netrin-1 plus BoNT/C1 for 0–30 min. While fluorescent vesicle clusters persist in controls (A), red fluorescence rapidly disappears upon treatment with Netrin-1 (B), indicating the occurrence of secretion events. Treatment with BoNT/C1 prevents the disappearance of red puncta in Netrin-1-treated neurons (C). On the bottom right, merged images showing the BODIPY label recorded separately in the red (high concentration) and green (low concentration) channels. The red spots show Golgi-derived vesicles or vesicle clusters, whereas the green fluorescence outlines the plasmalemma of the growth cone, labeled with small amounts of BODIPY. D, E, Percentage of average intensity of red fluorescent puncta in growth cones for control and Netrin-1-treated hippocampal neurons (normalized to 100% at the onset of treatment) (D). The average intensity of spots recorded in the red channel comes back to control levels after treatment with BoNT/C1 (E). F, Confocal images of hippocampal growth cones immunolabeled for DCC and TI-VAMP. A merged image showing colocalization of both proteins is shown at the bottom. G, Coimmunoprecipitation experiments in HEK293 cells transfected with pCMV6TI-VAMP and Sytx1AEGFP, pCMVDCC, or Friz2-HA DNAs. DCC immunoprecipitation results in coassociation with Sytx1A, visualized with anti-GFP antibodies, and with TI-VAMP (anti-TI-VAMP antibodies). The reverse immunoprecipitation with anti-TI-VAMP antibodies confirms DCC and Sytx1 coimmunoprecipitation. GFP immunoprecipitation of Sytx1 reveals coassociation with DCC and TI-VAMP proteins. Immunoblots show no coimmunoprecipitation of Friz-2 with DCC and the SNAREs Sytx1 and TI-VAMP. H, Coimmunoprecipitation experiments in E15 and adult brain lysates. Sytx1 and TI-VAMP immunoprecipitation yields coimmunoprecipitation of DCC in both samples. DCC and TI-VAMP immunoprecipitation yields coassociation of Sytx1. DCC and Sytx1 immunoprecipitation yields coassociation of TI-VAMP. Immunoprecipitations with anti-myc antibody were used as controls. I, J, Confocal images showing open-book preparations from the chicken spinal cord. The injection of the control pIRES-EGFP construct did not interfere with commissural axon pathfinding (I). Downregulation of TI-VAMP using in ovo RNAi (J) interfered with commissural axon navigation to and across the floor plate, with many fibers being arrested before (arrows) or within the floor plate (FP) (arrowheads). K, Quantification of the axon guidance phenotype in open-book preparations of embryonic chicken spinal cords. RNAi-mediated downregulation of TI-VAMP in chicken spinal cords (n = 139 injection sites in 17 embryos) yielded similar commissural pathfinding errors to those obtained with silencing of Sytx1 (n = 135 injection sites in 18 embryos). We observed strong phenotypes at 37.9% of dsTI-VAMP and 34.6% of dsStx-1, and weak phenotypes at 17.2 and 25.7% of injection sites, respectively. By contrast, electroporation of a pIRES plasmid expressing EGFP under the β-actin promoter (297 injection sites in 28 embryos; 60.1% normal and 21% weak phenotypes) did not differ significantly from untreated controls (6.9% strong phenotypes, 13% weak; n = 489 injection sites in 47 embryos). L–O, Schematic diagram summarizing a model for the regulation of Netrin-1-dependent exocytosis in growth cones. In a steady-state situation, the growth cones present a low release of exocytosis vesicles (L). Netrin-1 activation of DCC receptors result in ligand-dependent clustering of DCC/Sytx1 complexes in activated membrane domains (M). Here, the formation of a SNARE complex between Sytx1 and TI-VAMP proteins occurs, thereby promoting exocytosis of vesicles at DCC-activated domains (N), resulting in membrane expansion (O). Significant differences are labeled by asterisks (**p ≤ 0.001). Scale bars: A, 2 μm; F, 3 μm; I, 50 μm. Error bars indicate SEM.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Labeling, Fluorescence, Concentration Assay, Derivative Assay, Immunolabeling, Transfection, Immunoprecipitation, Western Blot, Injection, Construct, In Ovo, Electroporation, Plasmid Preparation, Expressing, Activation Assay

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Sytx1 coassociates with the DCC receptor in brain tissue and in transfected cells. A, DCC and Sytx1 immunoprecipitation of E15 and adult forebrain homogenates. DCC immunoprecipitation yields coimmunoprecipitation of Sytx1 in both samples (top panels). Sytx1 immunoprecipitation yields coassociation of DCC (bottom panels). Anti-βIII-tubulin antibodies were used as loading controls. B, DCC and Sytx1 immunoprecipitation assays do not result in coimmunoprecipitation of Egr1. Levels of Egr1 protein lysates are shown (bottom). C, DCC associates with A and B isoforms of Sytx1. Forebrain homogenates were immunoprecipitated and immunocomplexes were subjected to urea/SDS-PAGE. DCC coimmunoprecipitates two Sytx1 bands corresponding to Sytx1A and 1B (top panel). D, E, Coimmunoprecipitation experiments in HEK293 cells. DCC immunoprecipitation results in coassociation with Sytx1A, visualized either with anti-GFP (D) or anti-Sytx1 (E) antibodies. The reverse immunoprecipitation with anti-GFP or anti-Sytx1 antibodies also reveals DCC (D, E). Note that the efficiency of the immunoprecipitation with anti-GFP antibodies is consistently higher than when using anti-DCC antibodies, which yields low recovery of proteins in this condition. F, DCC affinity pull-down experiments with purified His-Sytx1A. Incubation with anti-DCC antibody reveals DCC in the beads coupled to His-Sytx1A, but not in control beads. G, Sytx1A was transcribed and translated in vitro in the presence of [35S]Met, and incubated with glutathione-Sepharose beads coupled to GST-DCCCYT or GST-MUNC18a. After SDS-PAGE, gels were exposed to a storage phosphor screen. [35S]Met-Sytx1A binds to DCCCYT (as well as to MUNC18a), but not to empty beads. Binding of [35S]Met-Sytx1A to DCCCYT is decreased in the presence of nonradioactive Sytx1A (Sytx1A*). H, Sensorgram showing binding between the GST-DCCCYT domain and His-Sytx1A. Increasing concentrations of His-Sytx1A were injected into a chip where the GST-DCCCYT was cross-linked; the interaction was recorded as SPR changes [in response units (RU)]. Increasing concentrations of His-Sytx1A yielded higher responses. I, Plot of the steady-state response (in RU) between GST-DCCCYT and a range of concentrations of His-Sytx1A (red) or between control GST and His-Sytx1A (black). No binding is detected when GST protein was cross-linked, whereas a saturable response is observed when GST-DCCCYT was immobilized. J, Pull-down experiments in which brain extracts (P0) were passed through Ni2+-affinity columns to which recombinant His-Sytx1A or His-Sytx1AH3TM proteins were coupled. Western blot analyses show coprecipitation of DCC with similar efficiencies in both cases. No coprecipitation of DCC is detected when extracts are incubated with control GST-GFP or GST-MUNC18a columns, whereas strong coprecipitation was observed with GST-DCCcyt. The high DCC signal in GST-DCCcyt samples is probably due to multimerization of the DCC receptor through the P3 domain. K, Sensorgram showing binding between the T1DCC peptide and purified His-Sytx1A. The peptide was immobilized as described in Material and Methods, and then increasing concentrations of His-Sytx1A were injected. Responses (in RU) increase at increasing concentrations of His-Sytx1A as a function of the time (in seconds). L, Plot of the steady-state response between T1 peptide and His-Sytx1A (red) or between a control peptide and His-Sytx1A (black). While no binding is detected when the control peptide is cross-linked, specific binding is observed when T1 peptide is immobilized.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Transfection, Immunoprecipitation, SDS Page, Purification, Incubation, In Vitro, Binding Assay, Injection, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: The DCC receptor does not coassociate with SNAP25, VAMP2, or Syntaxin 4. A, Coimmunoprecipitation experiments in brain lysates (E15 and adult). Whereas DCC and Sytx1 coimmunoprecipitate, the DCC receptor does not coassociate with SNAP25, VAMP2, or Sytx 4 in brain lysates. The Sytx antibody used for the immunoblot was an anti-Sytx1A antibody in all cases, except in the column immunoprecipitated with anti-Sytx 4 antibodies. Inputs are shown to the right. B, Coimmunoprecipitation experiments in HEK293 cells transfected with pCMVDCC and SNAP25-FLAG, VAMP2-FLAG or Sytx1A-EGFP DNAs. DCC immunoprecipitation results in coassociation with Sytx1A visualized with anti-GFP antibodies, but not with SNAP25 or VAMP2 (FLAG antibodies). The reverse immunoprecipitation with anti-FLAG antibodies did not reveal DCC coimmunoprecipitation.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Western Blot, Immunoprecipitation, Transfection

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Characterization of protein regions required for Sytx1A/DCC interaction. A, Diagram summarizing Sytx1A domains and the truncated Sytx1AEGFP chimeras generated. B, Expression of the several Sytx1AEGFP DNAs in HEK293 cells, showing that they generate proteins of the appropriate Mr between 65 and 27 kDa, as revealed by immunoblotting with anti-GFP antibodies. C, Coimmunoprecipitation experiments in HEK293 cells cotransfected with the distinct Sytx1AEGFP constructs together with pCMV or pCMVDCC. DCC immunoprecipitations (200 μg) were revealed by immunoblotting with anti-DCC or anti-GFP antibodies (left panel). Cells cotransfected with pCMVDCC and Sytx1AFLEGFP or Sytx1AH3TMEGFP show positive coimmunoprecipitation of Sytx1A fusions. The reverse immunoprecipitation assays with anti-DCC antibodies also show coimmunoprecipitation with DCC, exclusively when cells are cotransfected with Sytx1AFLEGFP or Sytx1AH3TMEGFP DNAs (arrows in top right panel). The bands corresponding to the distinct Sytx1AEGFP chimeras are labeled with asterisks in the bottom right panel label. D, Diagram summarizing the DCC domains and the truncated EGFPDCC chimeras generated. E, Western blot, revealed with an anti-GFP antibody, demonstrating appropriate Mr (between 90 and 40 kDa) of the distinct EGFPDCC chimeras expressed in HEK293 cells. F, Coimmunoprecipitation experiments in HEK293 cells cotransfected with the distinct EGFPDCC constructs together with pRcCMV or pRcCMVSytx1A DNAs. Sytx1 immunoprecipitations (200 μg) were revealed by immunoblotting with anti-GFP or anti-Sytx1 antibodies (left panel). All the cells cotransfected with pRcCMVSytx1A and the EGFPDCC constructs show coimmunoprecipitation of EGFP-tagged DCC chimeras (arrows), except when cells are cotransfected with EGFPP1-P3DCC DNA. The reverse immunoprecipitation assays with anti-GFP antibodies also reveal coimmunoprecipitation with Sytx1A (arrows in bottom right panel) in all cotransfected cells, except in those transfected with EGFPP1-P3DCC DNA. The bands corresponding to the distinct Sytx1AEGFP chimeras are labeled with asterisks in the top right panel.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Generated, Expressing, Western Blot, Construct, Immunoprecipitation, Labeling, Transfection

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Netrin-1, but not BDNF, triggers DCC mobilization and DCC/Sytx1 colocalization. A, Confocal images of hippocampal axonal shafts treated with Netrin-1-conditioned media for 0, 15, and 30 min, immunolabeled for DCC (red) and Sytx1A (green). Note that DCC and Sytx1A do not colocalize in axonal shafts in either condition. B, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0–30 min, immunolabeled for DCC and Sytx1. Note increased DCC/Sytx1 colocalization in growth cones incubated with Netrin-1. C, Quantification of DCC/Sytx1 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/Sytx1 colocalization over total Sytx1 signals) in cultures treated with Netrin-1-conditioned (black bars) or control-conditioned (white bars) media. D, Quantification of DCC/Sytx1 coimmunoprecipitation in hippocampal cultures treated with Netrin-1. Sytx1 immunoprecipitation reveals an increase in coassociated DCC in neurons treated with Netrin-1 (black bars). Immunoprecipitation with anti-DCC antibodies shows a marked increase in Sytx1 signals (white bars). E, Quantification of DCC/Sytx1 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/Sytx1 colocalization over total Sytx1 signal) in cultures treated with BDNF (gray bars) or control (white bars). Note that BDNF does not increase colocalization of DCC and Sytx1. F, Western blots from hippocampal cultures treated with BDNF for 15–30 min and immunoprecipitated with anti-DCC or anti-Sytx1A antibodies. Immunoblots reveal that BDNF does not increase the coassociation of DCC with Sytx1A. G, Western blots from hippocampal cultures treated with Netrin-1-conditioned (N) or control-conditioned (C) media for 0–30 min (left), and immunoprecipitated with anti-DCC (top panel) or anti-Sytx1 (bottom panel) antibodies. Immunoblots reveal increased association of DCC and Sytx1 in neuronal cultures treated with Netrin-1. Immunoprecipitations of brain lysates from newborn wild-type, heterozygous and homozygous netrin-1 mutant mice reveal decreased DCC/Sytx1 association in the null mutants (right). Note decreased coimmunoprecipitation of Sytx1 (top) and DCC (middle) in null-mutant brains. H, Quantification of DCC and Sytx1 coimmunoprecipitations in homogenates from newborn wild-type, heterozygous and homozygous netrin-1 mutant mice. Immunoprecipitations with either anti-DCC (white bars) or anti-Sytx1 (black bars) antibodies reveal a marked reduction in DCC/Sytx1 coassociation in netrin-1-null mutants. Significant differences are labeled by asterisks (*p ≤ 0.05; **p ≤ 0.001). Scale bar: A, B, 3 μm. Error bars indicate SEM.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Immunolabeling, Incubation, Immunoprecipitation, Western Blot, Mutagenesis, Labeling

Journal: The Journal of Neuroscience

Article Title: A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

doi: 10.1523/JNEUROSCI.3018-11.2011

Figure Lengend Snippet: Netrin-1 does not trigger coassociation of DCC with the SNAREs SNAP25 and VAMP2. A, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0 and 15 min, immunolabeled for DCC and the SNAREs SNAP25 and VAMP2. Note low colocalization signals of DCC and SNAP25 or VAMP2, both in control conditions and after incubation with Netrin-1. B, C, Quantification of DCC/SNAP25 and DCC/VAMP2 colocalization signals in hippocampal growth cones (expressed as percentage of DCC/SNARE colocalization over total SNARE signals) in cultures treated with Netrin-1-conditioned (black bars) or control-conditioned (white bars) media. Note decreased DCC/SNARE colocalization signals after Netrin-1 treatment. D, Western blots from hippocampal cultures treated with Netrin-1- (N) or control- (C) conditioned media for 0–30 min, and immunoprecipitated with anti-DCC or anti-Sytx1A antibodies. Immunoblots reveal no coimmunoprecipitation of DCC with the SNAREs SNAP25 and VAMP2 after incubation with DCC. Note coimmunoprecipitation of Sytx1A with SNAP25 and VAMP2. E–G, Confocal images of hippocampal growth cones treated with Netrin-1-conditioned media for 0 and 15 min, and in the presence of BoNT/C1. Cultures were immunolabeled for DCC and stained with phalloidin. Note that the mobilization of DCC to the axonal membrane after incubation with Netrin-1 (F) is not altered by BoNT/C1 incubation (G). H, Quantification of DCC signals in the periphery of and inside growth cones treated with Netrin-1-conditioned media for 0, 15, and 30 min, showing mobilization of DCC to the axonal membrane after incubation with Netrin-1. Whereas no DCC mobilization is detected after incubation with BDNF, treatment with BoNT/C1 does not alter DCC mobilization. I–L, Confocal images of control growth cones (I) and cones incubated with Netrin-1-conditioned media for 15 min (J), and with Netrin-1/BoNT/C1 (K). Cultures were immunolabeled for DCC (red) and Sytx1 (green). Note that the increase in DCC/Sytx1 colocalization in J is blocked after incubation with BoNT/C1 (K). L, Histograms illustrating DCC/Sytx1 colocalization in several experimental conditions. Significant differences are labeled by asterisks (*p ≤ 0.05, **p ≤ 0.001). Scale bar: A, 3 μm. Error bars indicate SEM.

Article Snippet: Western blot was performed using a monoclonal antibodies against DCC (BD Biosciences Pharmingen) and HRP-coupled rabbit anti-mouse as secondary antibody.

Techniques: Immunolabeling, Incubation, Western Blot, Immunoprecipitation, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Maintenance of Axo-Oligodendroglial Paranodal Junctions Requires DCC and Netrin-1

doi: 10.1523/JNEUROSCI.3285-08.2008

Figure Lengend Snippet: Disruption of the domain organization of the node of Ranvier in long-term netrin-1- and DCC-deficient slice cultures. Long-term (60 DIV) cerebellar slice cultures were double-labeled with antibodies against Na+ch and Kv1.2 (A–D). Kv1.2 protein was visualized using Alexa 488-conjugated secondary antibodies (green), and Na+ch proteins were visualized using Alexa 546-conjugated secondary antibodies (red). In cultures lacking netrin-1 (C) or DCC (D), a reduced distance between Na+ channels localized within the node of Ranvier and K+ channels normally localized to the juxtaparanodal region was detected. This decrease was primarily attributable to the apparent “leaking” of K+ channels into the paranode, and occasionally the node itself (C, D, arrowheads; E). In netrin-1−/−, but not DCC−/− slices, the length of the Na+ch-positive domain was increased relative to control (C, arrow; F). Magnification: 100× objective; digital zoom, 4. Scale bar, 2 μm. *p < 0.05, **p < 0.005. Error bars indicate SEM.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal anti-Caspr (University of California Davis NeuroMab; catalog #75-001), guinea pig polyclonal anti-Caspr, rabbit polyclonal anti-Caspr (gift from Dr. David Colman, McGill University, Montreal, Quebec, Canada) ( Svenningsen et al., 2003 ), mouse monoclonal anti-DCC intracellular domain (DCC IN ) (BD Biosciences Pharmingen; catalog #554223), rabbit polyclonal anti-Kv1.2 (Alomone Labs; catalog #APC-010), mouse monoclonal anti-myelin basic protein (MBP) (Millipore Bioscience Research Reagents; catalog #MAB382), rabbit polyclonal anti-MBP (Millipore Bioscience Research Reagents; catalog #AB9046), rat monoclonal anti-MBP (Millipore Bioscience Research Reagents; catalog #MAB386), rabbit polyclonal anti-netrin PN2 ( Manitt et al., 2001 ), rabbit polyclonal anti-neurofascin NFC2 (gift from Prof. Peter Brophy, University of Edinburgh, Edinburgh, UK; recognizes both 155 and 186 kDa isoforms of neurofascin) ( Tait et al., 2000 ), mouse monoclonal anti-neurofilament 145 kDa (NFM) (Millipore Bioscience Research Reagents; catalog #MAB1621), chicken polyclonal anti-NFM (EnCor Biotechnology; catalog #CPCA-NF-M), chicken polyclonal anti-neurofilament 200 kDa (NFH) (EnCor; catalog #CPCA-NF-H), mouse anti-myelin proteolipid protein (PLP) (Millipore Bioscience Research Reagents; catalog #MAB388), mouse monoclonal anti-sodium channel (pan) (Na + ch) (Sigma-Aldrich; catalog #S8809).

Techniques: Labeling

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Primer sequences

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques: Sequencing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Antibodies used in immunohistochemistry and Western blot experiments

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques: Immunohistochemistry, Western Blot, Incubation

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Amplification of netrin-1 and its receptor cDNAs. Gel electrophoresis and ethidium bromide staining of netrin-1 (Lane 1), UNC5B (Lane 2), and DCC (Lane 3) RT-PCR products from purified cytotrophoblast cells. MW, molecular weight marker.

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques: Amplification, Nucleic Acid Electrophoresis, Staining, Reverse Transcription Polymerase Chain Reaction, Purification, Molecular Weight, Marker

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Western blot analysis of the netrin-1 receptor DCC. Western blot analysis of DCC was performed with 100 μg protein extracts, except for Lanes 5 and 6, which were performed with 70 μg and 35 μg, respectively. The 200-kDa band is the DCC receptor; the strong band at 120 kDa is a proteolytic fragment of the receptor. (A) Lane 1, at-term placenta tissue extract; Lane 2, trophoblast tissue extract (10 weeks gestation); Lane 3, 48-hr-cultured cytotrophoblasts; Lane 4, colon used as a positive control; Lane 5, 48-hr-cultured cytotrophoblast protein extract (70 μg); and Lane 6, 48-hr-cultured cytotrophoblast protein extract (35 μg). Lanes 1–4 are from one experiment; Lanes 5 and 6 are from another experiment. The Western blots are representative of six separate experiments, each performed with samples prepared in different experiments and used only once. (B) Closer view of DCC antibody-labeled bands in the 200-kDa area in 48-hr-cultured cytotrophoblasts, 10 weeks placenta, and colon.

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques: Western Blot, Cell Culture, Positive Control, Labeling

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Immunohistology of netrin-1, UNC5B, and DCC in first-trimester human placental tissue. (A–F) Histological sections of anchoring villus. (G–I) Histological sections of decidua. Immunolabeling was as follows: (A) Goat anti-netrin-1; immunoreactivity was found in villous and extravillous cytotrophoblasts and in external surface of syncytiotrophoblasts. (B,I) Mouse anti-DCC showing immunoreactivity in syncytiotrophoblasts and in extravillous cytotrophoblasts located at decidua. (C) Goat anti-UNC5B showing immunoreactivity in villous cytotrophoblasts and in extravillous cytotrophoblasts located proximally to anchoring villus. (D,G) Anti-rabbit c-ErbB2 showing immunoreactivity in extravillous cytotrophoblasts and in external surface of syncytiotrophoblasts. (E,H) Anti-mouse cytokeratin 7 showing immunoreactivity in villous and extravillous cytotrophoblasts and in syncytiotrophoblasts. (F) Negative control processed in the absence of primary antibody.

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques: Immunolabeling, Negative Control

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Immunohistochemical localization of netrin-1, UNC5B, and DCC in term human placental tissue. (A–G) Serial histological sections in decidua. (H) Section in villous trophoblasts. Small arrows indicate decidual cells, and large arrows indicate invasive extravillous cytotrophoblasts. Immunohistology of at-term human placental tissue for netrin-1, UNC5B, and DCC and the relevant c-ErbB2, vimentin, and cytokeratin 7 controls were as follows: (A) Anti-rabbit c-ErbB2 immunoreactivity in extravillous cytotrophoblasts. (B) Anti-mouse cytokeratin immunorectivity in extravillous cytotrophoblasts. (C) Negative control processed in the absence of primary antibody. (D) Anti-vimentin immunoreactivity in decidual cells. (E) Anti-goat UNC5B immunoreactivity in decidual cells and weak staining in extravillous cytotrophoblasts. (F) Anti-mouse DCC immunoreactivity in extravillous cytotrophoblasts and (H) in syncytiotrophoblasts. (G) Anti-goat netrin-1 immunoreactivity in decidual cells, and (I) in syncytiotrophoblasts.

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques: Immunohistochemical staining, Negative Control, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Characterization and Expression of Netrin-1 and Its Receptors UNC5B and DCC in Human Placenta

doi: 10.1369/jhc.2009.953463

Figure Lengend Snippet: Distribution of netrin-1 and its receptors in first-trimester and term placentas

Article Snippet: After rinsing the slides in buffer for 5 min, slides were incubated with the commercial primary antibodies (see ) polyclonal goat anti-N-terminal netrin-1 (dilution 1:50), rabbit anti-C-terminal netrin-1 (dilution 1:50), goat anti-UNC5B (dilution 1:50) for 30 min, or monoclonal mouse anti-human DCC antibody (BD Pharmingen; San Diego, CA, diluted 1:100) for 40 min at 37C.

Techniques:

Journal: Neural Development

Article Title: Netrin-1 directs dendritic growth and connectivity of vertebrate central neurons in vivo

doi: 10.1186/s13064-015-0041-y

Figure Lengend Snippet: Specific patterns of DCC and UNC-5 expression in the X. laevis central nervous system. Immunostaining with antibodies to UNC-5 ( red ) and DCC ( green ) revealed specific patterns of expression of the netrin-1 receptors in stage 45 tadpoles. a–g UNC-5 ( red ) and DCC ( green ) immunoreactivity in the forebrain ( a ), pre-tectum ( b ), caudal tectum ( e ), hindbrain ( f ), and rostral spinal cord ( g ) demonstrate a specific pattern of expression for each of these receptors within subpopulations of neurons in the central nervous system. c UNC-5 immunostaining ( red ) localizes to subpopulations of neurons in the dorsal tectum, lateral-ventral midbrain, ventral midline ( vm ), and infundibulum ( if ). d DCC immunoreactivity ( green ) is localized in dorsal tectal neuron cell bodies and processes in the tectum and ventral midline, as well as in the tectal neuropil ( np ). e, f Note the specificity of immunostaining and co-localization of UNC-5 and DCC expression in subpopulations of cells in the caudal tectum ( e ) and hindbrain ( f ) and the localization of DCC receptors to discrete fiber tracts ( arrows ). g, h UNC-5 ( red ) and DCC ( green ) immunoreactivity in the rostral ( g ) and caudal ( h ) spinal cord is localized to fiber tracts and ventral midline in agreement with published observations in Xenopus and other species (for review, see [ , , – ]). DCC immunoreactivity in the spinal cord is similar when staining with antibodies directed against the extracellular ( g ) or intracellular ( h , bottom) domains of DCC. Counterstaining with DAPI ( blue ) serves to distinguish nuclear staining from UNC-5 ( red ) and DCC ( green ) expression in cell bodies and fiber tracts. Scale bars: 50 μm

Article Snippet: Coronal and horizontal sections at the level of the optic tectum were incubated with the following primary antibodies without antigen retrieval step [ ]: mouse monoclonal antihuman presynaptic protein SNAP-25 (1:500 dilution; Enzo Life Science, Farmingdale, NY, USA), mouse monoclonal antibody against the extracellular domain of human DCC (1:100 dilution; anti-DCC, Genetex Clone AF5, Irvine, CA, USA), mouse monoclonal antibody against the intracellular domain of human DCC (1:1500 dilution; BD Biosciences Pharmingen, San Jose, CA, USA), chicken polyclonal antibody against human netrin-1 (1:3500, Novus Biologicals, Littleton, CO, USA), and rabbit polyclonal anti-mouse UNC-5H3 antibody (1:14,000 dilution; generous gift of Dr. Antony Pawson).

Techniques: Expressing, Immunostaining, Staining