Journal:

Article Title: Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

doi: 10.1093/emboj/20.20.5791

Figure Lengend Snippet: Fig. 1. Structural map of WRN protein motifs and recombinant proteins used in this study. (A) Conserved RecQ ATPase/helicase and exonuclease motifs, RecQ C-terminal region (RQC), helicase-related domain (HRDC), nuclear localization sequence (NLS) and known regions for WRN protein interactions are designated. Positions of site-directed mutations in motif I of the helicase domain (WRN-K577M) and motif I of the exonuclease domain (WRN-E84A) are indicated by asterisks. GST–WRN recombinant proteins are shown with subscripted numbers to designate the WRN amino acid sequences. (B) Purified full-length recombinant proteins (1 µg) and GST–WRN recombinant fragments (2–4 µg) used in this study were resolved on 10% polyacrylamide SDS gels run and stained with Coomassie Brilliant Blue. The 66 kDa band in the WRN protein preparations is BSA (100 µg/ml) (Orren et al., 1999). Bands migrating below GST–WRN949–1432 (∼85 kDa) were determined to be degradation products by western blot analysis using anti-GST antibody (Santa Cruz).

Article Snippet: Recombinant GST–WRN fusion proteins, overexpressed in E.coli , were purified on GS beads (Amersham Pharmacia Biotech) as recommended by the manufacturer (Figure B).

Techniques: Recombinant, Sequencing, Purification, Staining, Western Blot

Journal:

Article Title: Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

doi: 10.1093/emboj/20.20.5791

Figure Lengend Snippet: Fig. 2. WRN and FEN-1 interact physically. (A) Beads with GST (lane 2), GST–WRN949–1432 (lane 3) or GST–WRN1072–1236 (lane 4) were incubated with 750 µg of HeLa NE. In control experiments, NE was omitted during binding with GST–WRN949–1432 (lane 5) or GST–WRN1072–1236 (lane 6). Western blotting was performed with anti-FEN-1 antibodies. The NE input (lane 1) corresponds to 10 µg. (B) Beads with GST (lane 1) or GST–WRN949–1432 (lane 2) were incubated with 1.2 µg of recombinant FEN-1 purified from E.coli. In control experiments, FEN-1 was omitted during binding with GST–WRN949–1432 (lane 3). Western blotting was performed with anti-FEN-1 antibodies. The FEN-1 input (lane 4) corresponds to 20 ng. (C) FEN-1–Sepharose specifically binds recombinant WRN. Purified WRN and FEN-1 were judged to be pure of DNA by analysis using SYBR Green Stain (FMC Products). Western blot of the third wash (lanes 1 and 2) or eluted fractions (lanes 3 and 4) from binding reaction of WRN to FEN-1–Sepharose (lanes 2 and 4) or BSA–Sepharose (lanes 1 and 3) is shown. (D) WRN and FEN-1 coprecipitate from HeLa cell lysate using anti-WRN antibody as demonstrated by western blotting. Top panel, blot was probed with anti-FEN-1 antibody. Bottom panel, blot was probed with anti-WRN antibody. Lane 1, control precipitate from HeLa cell lysate in which WRN antibody was omitted from immunoprecipitation; lane 2, HeLa cell lysate input; lane 3, immunoprecipitate from HeLa cell lysate using WRN antibody; lane 4, AG11395 (WS–/–) cell lysate input; lane 5, immunoprecipitate from AG11395 cell lysate using WRN antibody; lane 6, purified His-tagged FEN-1, which migrates slightly higher than NE FEN-1.

Article Snippet: Recombinant GST–WRN fusion proteins, overexpressed in E.coli , were purified on GS beads (Amersham Pharmacia Biotech) as recommended by the manufacturer (Figure B).

Techniques: Incubation, Binding Assay, Western Blot, Recombinant, Purification, SYBR Green Assay, Staining, Immunoprecipitation

Journal:

Article Title: Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

doi: 10.1093/emboj/20.20.5791

Figure Lengend Snippet: Fig. 7. A C-terminal fragment of WRN retains the ability to stimulate the FEN-1 cleavage reaction. Reactions (20 µl) containing 10 fmol of 1 nt 5′ flap DNA substrate and the indicated amounts of FEN-1 were incubated at 37°C for 15 min under standard conditions. The reactions in the presence of GST–WRN fragments contained 75 fmol of WRN fragment. (A) Phosphorimage from a typical gel. Lane 1, no enzyme; lane 2, GST–WRN949–1432; lane 3, 5 fmol of FEN-1; lane 4, GST–WRN949–1432 + 5 fmol of FEN-1; lane 5, 5 fmol of FEN-1 + GST–WRN1072–1236; lane 6, 10 fmol of FEN-1; lane 7, 10 fmol of FEN-1 + GST–WRN949–1432; lane 8, 10 fmol of FEN-1 + GST–WRN1072–1236; lane 9, 20 fmol of FEN-1; lane 10, 20 fmol of FEN-1 + GST–WRN949–1432; lane 11, 20 fmol of FEN-1 + GST–WRN1072–1236. (B) % incision (mean value of three experiments) with SD. Filled circles, FEN-1; open circles, FEN-1 + GST–WRN949–1432; open squares, FEN-1 + GST–WRN1072–1236.

Article Snippet: Recombinant GST–WRN fusion proteins, overexpressed in E.coli , were purified on GS beads (Amersham Pharmacia Biotech) as recommended by the manufacturer (Figure B).

Techniques: Incubation

Journal:

Article Title: Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

doi: 10.1093/emboj/20.20.5791

Figure Lengend Snippet: Fig. 8. Mapping of the FEN-1 interaction domain that mediates the functional interaction between WRN and FEN-1. Reactions (20 µl) containing 10 fmol of 1 nt 5′ flap DNA substrate, 10 fmol of FEN-1 and 75 fmol of the indicated GST–WRN fragment were incubated at 37°C for 15 min under standard conditions. (A) Phosphorimage of a typical gel. Lane 1, no enzyme; lane 2, FEN-1; lane 3, FEN-1 + GST–WRN949–1432; lane 4, FEN-1 + GST–WRN949–1236; lane 5, FEN-1 + GST–WRN1072–1236; lane 6, FEN-1 + GST–WRN949–1092; lane 7, FEN-1 + GST–WRN239–499; lane 8, GST–WRN949–1432; lane 9, GST–WRN949–1236; lane 10, GST–WRN1072–1236; lane 11, GST–WRN949–1092; lane 12, GST–WRN239–499. (B) % incision (mean value of three experiments) with SD.

Article Snippet: Recombinant GST–WRN fusion proteins, overexpressed in E.coli , were purified on GS beads (Amersham Pharmacia Biotech) as recommended by the manufacturer (Figure B).

Techniques: Functional Assay, Incubation

Journal: Genes & Development

Article Title: Histone H3K4 methylation regulates deactivation of the spindle assembly checkpoint through direct binding of Mad2

doi: 10.1101/gad.278887.116

Figure Lengend Snippet: Mad2 binds H3 methylated at K4 in a conformation-specific manner. ( A , C ) Colloidal staining of purified recombinant proteins. Pull-down assays of the indicated recombinant proteins to identify direct binding with calf thymus histones, assayed using immunoblots of membranes probed with antibodies against H2A, H2B, or H3. ( B , D ) Pull-down assay with recombinant GST-Mad2 fusion proteins and unmodified H3 (K4me0) or MLA full-length histones generated to mimic H3K4 monomethylation (K4me1), H3K4 dimethylation (K4me2), or H3K4 trimethylation (K4me3). Direct binding was assayed using immunoblots of membranes probed with an antibody recognizing histone H3. ( E,F ) GST pull-down assays using recombinant GST-yMad2 and GST-hMad2 with wild-type histone H3 or H3 with various point mutations. Direct binding was assessed by using immunoblots of membranes probed with an antibody against H3.

Article Snippet: Similar to the calf thymus histone-binding assay, 2 μg of recombinant GST fusion proteins was incubated with 1 μg of full-length recombinant histone H3 with either no methylation on K4 or methylated lysine analogs (K4me1, K4me2, and K4me3) (Active Motif).

Techniques: Methylation, Staining, Purification, Recombinant, Binding Assay, Western Blot, Pull Down Assay, Generated

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: Primary Antibodies

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Affinity Purification, Purification, Recombinant

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a-l) triple-label immunofluorescence of HEK293 cells transfected with Pcdh-γC4-mCherry (a-c); mCherry (d-f); Pcdh-γC3-EGFP (g-i) or Pcdh-γC5-EGFP (j-l). Panels a and d show mCherry fluorescence (magenta). Panels g and j show EGFP fluorescence (green). The Rb anti-Pcdh-γC4 antibody (green in b and e or magenta in h and k) recognizes Pcdh-γC4-mCherry (b) but not mCherry (e), Pcdh-γC3-EGFP (h) or Pcdh-γC5-EGFP (k). A Ms anti-Pan γ mAb recognizes the thee C-type Pcdh-γs (blue c, i and l) but not mCherry (f). Scale bar = 20 μm.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence, Transfection, Fluorescence

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a-d) HEK293 cells cotransfected with Pcdh8 and mCherry (magenta). Panels a and b (same cells) show that anti-Pcdh-γC4 Ab (green) does not recognize cells co-transfected with Pcdh8 and mCherry (arrowhead). In contrast, co-transfected cells are recognized by the anti-Pcdh8 antibody (c, green). Panel d shows the absence of green Pcdh8 immunofluorescence in a non-transfected cell (not expressing mCherry). (e-h) HEK293 cells transfected with Pcdh-γC4-mCherry. Panels e and f show that the anti-Pcdh-γC4 antibody (e, green) reacts with the transfected cell (f, mCherry magenta). However, the anti-Pcdh8 Ab (green) does not react (g and h) with the transfected cell (arrowhead). Scale bar = 20 μm.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Transfection, Immunofluorescence, Expressing

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: Triple-label immunofluorescence with Rb anti-Pcdh-γC4 (green), Ms anti-Pcah8 (blue) and GP anti-VGAT (magenta). (a and b) are the same field of Purkinje cell layer (PK), molecular layer (ML) and granule cell layer (GR) of the cerebellum. The asterisk marks a Purkinje cell and the arrow the corresponding GABAergic pinceaux in the AIS (magenta). (c) ML layer of the cerebellum. While Pcdh-γC4 immunofluorescence concentrates in the Bergmann glia processes (green, vertical alignments), Pcdh8 immunofluorescence (blue) profusely labels the neuropil of the ML. (d-e) olfactory bulb. GL indicates an olfactory glomerulus. Asterisks indicate some periglomerular neurons. Arrowheads point to co-localization of VGAT-terminals (magenta) with Pcdh8 (blue). Pcdh-γC4 puncta (green) are adjacent to VGAT-terminals. There is little co-localization between Pcdh-γC4 and Pcdh8. Scale bar = 10 μm.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a) Rat forebrain membranes from P37 show three main protein bands of 120, 55 and 40 kD. The immunoreaction with the three proteins is blocked with 20 μg/ml of peptide antigen (strip P). They are two nitrocellulose strips from the same transfer processed in parallel, under the same conditions. The mobility of Mw markers (in kD) is shown at the left. (b) Comparative expression of the 120 kD Pcdh-γC4 protein as revealed with anti-Pcdh-γC4 (green). The left lane shows the mobility of Mw protein markers (150 and 100 kD, magenta). Next two left lanes show P0 cleared brain homogenates of wild type (WT) and homozygous (HZ) mutant TCKO mouse. Right side lanes show cleared homogenates of E18 and P0 rat brains. Underneath (magenta) shows the immunoblot of the same lanes with anti-β-actin antibody, revealing a 42 kD β-actin protein band that migrates between the 50 and 37 kD protein markers, (left lane). (c) The 120 kD Pcdh-γC4 protein (green) is expressed in the P30 and P45 rat brain cleared homogenates. Also, note the enrichment of the 120 kD Pcdh-γC4 protein in the membranes of the P37 rat brain. (d) Developmental expression of the 120 kD Pcdh-γC4 protein in the rat brain. The left lanes show the WT and HZ TCKO mice, for protein band identification. Underneath, in magenta, are the same lanes with anti-β-actin. Same amount of total protein was added in each lane (40 μg).

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Stripping Membranes, Expressing, Mutagenesis, Western Blot

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: Rat hippocampal cultures. (a-d) Double-label immunofluorescence with Rb anti-Pcdh-γC4 (γC4, green) and sheep anti-GAD (blue). Arrowheads show Pcdh-γC4 puncta at contact points between an incoming axon and dendrites. Filled arrowheads show Pcdh-γC4 puncta associated with GAD+ boutons. Empty arrowheads show Pcdh-γC4 puncta associated with axon-neuron contacts but not boutons. Panels a and b show the overlays. Note than in a and c the GABAergic axon (blue) contacts a dendrite of a GABAergic interneuron (blue, right side of the panel) and a pyramidal neuron (left side of the panel). Panels b and d show an axon (blue) contacting a dendrite of a GABAergic interneuron (blue). Note that Pcdh-γC4 puncta associated or not associated with the axon are larger in the contacts with interneurons in a and b, than in the contacts with the pyramidal neuron in a. (e-g) Triple label immunofluorescence with Rb anti-Pcdh-γC4 (green), sheep anti-GAD (blue) and Ms anti-gephyrin (magenta). Some Pcdh-γC4 puncta are localized at contact points between the axon and neuron (arrowheads), however, the majority of these Pcdh-γC4 puncta are adjacent to, but seldom co-localize with (arrow), gephyrin clusters. (h-j) Triple label immunofluorescence with Rb anti-Pcdh-γC4 (green), GP anti-VGLUT1 (blue) and Ms anti- PSD-95 mAb (magenta). Some Pcdh-γC4 puncta are associated with glutamatergic synapses (arrowheads) but the majority of Pcdh-γC4 puncta are adjacent to, but do not co-localize with VGLUT1 puncta or PSD-95 clusters. Scale bar = 10 μm.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a-c) Double-label immunofluorescence with the Rb anti-Pcdh-γC4 (γC4, magenta) and Ms anti-pan-Pcdh-γ (Panγ, green). Panel c shows the overlay (Over). (d-i) Double-label immunofluorescence with the Rb anti-Pcdh-γC4 (green) and sheep anti-GAD (blue). Panels d and e show GAD-glutamatergic pyramidal neurons. Panels f-i show GAD+ GABAergic interneurons. Arrows point to very large Pcdh-γC4 puncta present in some interneurons. (j-k) Immunofluorescence with Rb anti-Pcdh-γC4 (γC4, green) of non-permeabilized surface-labelled (j) and permeabilized (k) neurons from the same experiment. Arrowheads point to Pcdh-γC4 puncta in surface-labeled neurons. Arrows point to large Pcdh-γC4 puncta. Scale bar = 20 μm for a-i and 10 μm for j-k.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence, Labeling

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: Triple-label immunofluorescence with Rb anti-Pcdh-γC4 (green), GP anti-VGAT (magenta) and Ms anti-S100β (blue). (a) Glomerular layer of the olfactory bulb. Pcdh-γC4 puncta are present at the contacts (arrowheads) between periglomerular astrocytes (blue) and periglomerular neurons (asterisks). Pcdh-γC4 puncta are also present inside the glomerulus. (b) Granule cell layer of the olfactory bulb. Pcdh-γC4 puncta concentrate on contacts (arrowheads) between astrocytes (soma and processes) and neurons, frequently apposed to VGAT-containing terminals. Note the row of granule neurons (asterisks). Pcdh-γC4 puncta are largely absent from the contacts granule cell to granule cell. (c) External plexiform layer of the olfactory bulb. Pcdh-γC4 puncta are frequently associated with astrocyte processes and VGAT puncta (arrowheads). (d) Cerebellum. The abbreviations for the layers are as defined in the legend to Fig 10. Note the presence of Bergmann glia cell bodies on the PK layer (arrow), their radial fibers in the ML and the associated Pcdh-γC4 puncta (arrowheads). (e) Purkinje cells, also shown in d, are extensively surrounded by Bergmann glia (blue). There are Pcdh-γC4 puncta in the contacts between these Purkinje cells and Bergmann glia (arrowheads). Pcdh-γC4 puncta also associate with the VGAT-containing pinceaux (magenta, asterisk) and astrocyte processes in the AIS of the Purkinje cell. (f) Radial Bergmann glia fibers in the ML of the cerebellum have associated Pcdh-γC4 puncta, often associated with VGAT terminals. (g) Granule cell layer of the cerebellum. Note the astrocytic processes (blue) surrounding the VGAT-containing synaptic glomeruli (magenta) and the Pcdh-γC4 puncta (green) associated with the synaptic glomeruli (arrows). Pcdh-γC4 puncta are also present in the contacts between astrocyte processes and granule cells (arrowheads). Scale bar = 10 μm in a; 11.5 μm in b; 5.5 μm in c; 20 μm in d; 13 μm in e and f; and 17 μm in g.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a, c and e) glomerular layer of olfactory bulb. (b and d) granule cell layer of olfactory bulb. (f) External plexiform layer of olfactory bulb. (g and h) cerebellum. All panels show immunofluorescence with Rb anti-Pcdh-γC4 (magenta) and GP anti-VGLUT1 (blue). Panels a, b and f also show Ms PSD-95 immunofluorescence (green). Boxed areas in a, b and c are shown at higher magnification in c, d and e respectively. Panel a is mostly occupied by an olfactory glomerulus labeled OG in the center. The asterisk in a and c are placed in the same position inside the OG to serve as a reference. The arrows in a and c point to the same VGLUT1 puncta inside the glomerulus, which are also associated with Pcdh-γC4 puncta. Arrowheads in e point to Pcdh-γC4 puncta associated with VGLUT1 terminals. Numbers in b and d indicate some granule cell aggregates in the olfactory granule layer. (g) In the cerebellum, the arrow indicates the region with the highest concentration of bright Pcdh-γC4 puncta, which is at the boundary between Purkinje cell layer (PK) and granule cell layer (GR). Arrowheads point to a row of Pcdh-γC4 puncta in the molecular layer (ML). (h) Granule cell layer of the cerebellum. Asterisks indicate rows of granule cells. Note the absence of Pcdh-γC4 puncta in-between granule cells of the same row and the presence of Pcdh-γC4 puncta (arrowheads) separating VGLUT1 labeled synaptic glomeruli (blue) and granule cells. Scale bar represents 20 μm for a and b; 10 μm for c and d; 5 μm for e and f; and 12 μm for g and h.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence, Labeling, Concentration Assay

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a-h) Double-label immunofluorescence with Rb anti-Pcdh-γC4 (green) and GP anti-VGAT (blue) in cerebellum (a), dentate gyrus (b) and layer III of cerebral cortex (c-h). Numbers indicate somas of some individual neurons. In a and b, the abbreviations for the layers are as defined in the legend to Fig 10. Arrowheads in a point to Pcdh-γC4 puncta associated with the soma of PK cells. Arrows in a point to Pcdh-γC4 puncta associated with the VGAT-containing pinceaux contacting the AIS of Purkinje cells. Crossed arrowheads in a point to Pcdh-γC4 puncta associated with VGAT-enriched synaptic glomeruli in the granule cell layer. Arrowheads in a, d, e, g and h point to Pcdh-γC4 puncta that are adjacent to VGAT terminals. (i and j) Triple-label immunofluorescence with Rb anti-Pcdh-γC4 (green) and GP anti-VGAT (blue) and Ms Geph (magenta) in layer III of the rat cerebral cortex. Arrowheads in i and j point to Pcdh-γC4 puncta that are adjacent to GABAergic synapses. (k-m) Double-label immunofluorescence with Rb anti-Pcdh-γC4 (magenta) and Ms anti-S100β (green). Arrowheads in l and m point to Pcdh-γC4 puncta associated with S-100β-labeled astrocyte processes. The symbol # indicates blood vessels in c and k. Boxed areas in d, g and l are shown at higher magnification in e, h and the left side of m respectively. Scale bar represents 10 μm for a, b, d, g and l; 20 μm for c, f and k; 5 μm for e, h and m; and 4.4 μm for i and j.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence, Labeling

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: Double-label immunofluorescence. Boxed areas are shown at higher magnification underneath the corresponding panel. (a-f) double-label immunofluorescence of WT mouse neurons with the Rb anti-Pcdh-γC4 (green) and Ms pan-Pcdh-γA mAb (magenta). Panels c and f show the corresponding overlays (Over). Arrows show co-localization of Pcdh-γC4 and pan-Pcdh-γA puncta. Filled arrowheads point to Pcdh-γC4 puncta with no corresponding pan-Pcdh-γA puncta. Empty arrowheads show pan-Pcdh-γA puncta with no corresponding Pcdh-γC4 puncta. (g-l) TCKO mouse neurons do not have Pcdh-γC4 puncta (green) but have pan-Pcdh-γA puncta (magenta). Panels i and l show the corresponding overlays (Over). The scale bar represents 20 μm for a-c and g-i; and 8 μm for d-f and j-l.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a) Immunocytochemistry with anti-Pcdh-γC4 of a parasagittal section. (b) CA3 region of the HP. (c) Higher magnification of the boundary between the stratum pyramidale (SP) and stratum radiatum (SR) of the CA3 region of the HP. Note the granular aspect of the immunoreaction in the SP layer. (d) Olfactory glomeruli (OG) of the olfactory bulb. Asterisks indicate individual olfactory glomeruli. Arrowheads point to strong immunoreaction at the boundary surrounding the glomeruli (e) Dentate gyrus. (f) Higher magnification of the boundary between granule cell layer (GR) and the plexiform layer (PL) of the dentate gyrus. Note the granular aspect of the concentration of immunoreaction in the boundary. (g) Cerebral cortex, layers II-IV. (h) Cerebellum. (I and j) Higher magnification of the boundary between the Granule cell layer (GR) and Purkinje cell layer (PK). Granular accumulations of the immunoreaction occur in the GR layer (white arrowheads), in the boundary between Purkinje cells and the GR layer (black arrowheads) and surrounding blood vessels (black arrow). Abbreviations: CB, cerebellum; CC, cerebral cortex; St, corpus striatum; DG, dentate gyrus; GR, granule cell layer; HL, hilus; HP, hippocampus; ML, molecular layer; OB, olfactory bulb; OG, olfactory glomerulus; Pn, pontine nuclei; PL, plexiform layer; PK, Purkinje cell layer; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SN, substantia nigra. Scale bar = 5 mm in a; 1 mm in b, e and h; 400 μm in g; 200 μm in d; and 100 μm in c, f, I and j.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunocytochemistry, Concentration Assay

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: Triple-label immunofluorescence with Rb anti-Pcdh-γC4 (green), Ms anti-GFAP mAb (magenta) and DAPI (blue). Astrocytes with different morphologies are identified by anti-GFAP immunofluorescence. (a-g) Pcdh-γC4 puncta concentrate around the nucleus (a, b, d and f). Boxed areas in b, d and f are shown at higher magnification in c, e, and f respectively. Pcdh-γC4 immunofluorescence also associates with the plasma membrane (e) and with astrocyte processes (c and g). Scale bar represents 20 μm for a, b, d and f; and 10 μm for c, e and g.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence

Journal: The Journal of comparative neurology

Article Title: Expression of Protocadherin-γC4 protein in the rat brain

doi: 10.1002/cne.24783

Figure Lengend Snippet: (a and c) triple-label immunofluorescence with Rb anti-Pcdh-γC4 (magenta), Ms anti-S100β (blue) and Chicken anti-GFP (green) of the cerebral cortex of a P35 rat that had been in utero electroporated with EGFP. The EGFP immunofluorescence (green) reveals two transfected pyramidal neurons of layer III expressing EGFP. Astrocyte processes are identified by S100β immunofluorescence (blue). (b and d) correspond to a and c respectively, in which the magenta channel has been eliminated to better appreciate the localization of the Pcdh-γC4 puncta (arrowheads) at contacts between astrocyte processes (blue) and pyramidal neurons (green). Scale bar = 10 μm.

Article Snippet: summarizes the primary antibodies used in this communication. table ft1 table-wrap mode="anchored" t5 Table I. caption a7 Antigen Immunogen Manufacturer Dilution Pcdh-γC4 Synthetic peptide (DHAPRFPRQQLDLE) AA 128–141 of rat Pcdh-γC4 Made in our laboratory, Rabbit polyclonal 1:150 1 1:30 to 1:50 2 1:600 3 1:100 4 1:25 5 Pan-Pcdh-γ AA 808–931 (C-terminal constant domain) of mouse Pcdh-γA1 NeuroMab, Cat# 73–185, Lot# 437–3VA-24, RRID:AB_10676098; mouse monoclonal, clone N159/5.

Techniques: Immunofluorescence, In Utero, Transfection, Expressing