Journal: Nature neuroscience

Article Title: GLIAL ANKYRINS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY

doi: 10.1038/nn.3858

Figure Lengend Snippet: No paranodal or axonal abnormalities were observed in sciatic nerves of AnkB-cKO mice. ( a–l ) Immunostaining of sciatic nerve longitudinal sections from 4 to 6-week-old ( a–i, l ), P3 ( j , AnkB, N105/17) and 1-year-old ( k ) mice shows normal paranodal and juxtaparanodal domains and proper Nav channel subtype switch in AnkB-cKO mice. NFasc, neurofascin (both nodal 186-kDa and paranodal 155-kDa isoforms were stained). Cntn, contactin. βIV, βIV spectrin. ZO-1, zona occludens protein 1. MAG, myelin-associated glycoprotein. CNP, 2′,3′-cyclic nucleotide 3′ phosphodiesterase. Necl4, nectin-like protein 4. Two mice per genotype and more than 75 ( j ) or 150 ( a–i, k, l ) nodes were examined in each mouse. Scale bars = 5 μm ( a, b; c–g; h; i; j; k; l ).

Article Snippet: The primary antibodies used were: rabbit antibodies against AnkB membrane-binding domain , AnkB C-terminal region (H-300, SantaCruz), AnkG spectrin-binding domain and C-terminal region , AnkG exon 1b and exon 1e (generated by immunizing against peptides unique to these domains followed by affinity purification of specific antibodies), AnkG 480/270 (generated, affinity purified, and kindly provided by Dr. Paul Jenkins, Duke University), AnkR (generated against His-tagged, full-length human AnkR and affinity purified), GFP (A11122, Life Technologies), NF-M (AB1987, EMD Millipore), Caspr , βIV spectrin SD , Kv1.2 , Caspr2 (ab33994, Abcam), Nav1.2 , Nav1.6 , and 4.1G (kindly provided by Dr. Elior Peles); mouse monoclonal antibodies against AnkB membrane-binding domain (N105/13 and N105/17, UC Davis/NIH NeuroMab Facility), AnkB spectrin-binding domain (2.20, SantaCruz), AnkG spectrin-binding domain or C-terminal region (N106/36 and N106/65, NeuroMab), Caspr (K65/35, NeuroMab), contactin (K73/20, NeuroMab), actin (C4, EMD Millipore), ZO-1 (ZO1-1A12, Life Technologies), E-cadherin (4A2C7, Life Technologies), Kv1.2 (K14/16, NeuroMab), Nav1.6 (K87A/10, NeuroMab), CNP (11-5B, Sigma-Aldrich), MAG (513, EMD Millipore), MBP (SMI-94, Covance), Necl4 (N244/5, NeuroMab), pan-Nav channels (K58/35 ) and GFP (N86/38, NeuroMab); the rat monoclonal antibody against MBP (MAB386, EMD Millipore); chicken antibodies against NFasc (AF3235, R&D Systems) and βIV spectrin SD ; goat antibodies against contactin (AF904, R&D Systems), connexin 32 (C-20, Santa Cruz), and AnkG (generated against the C-terminal domain of AnkG minus the death domain and affinity purified).

Techniques: Immunostaining, Staining

Journal: Nature neuroscience

Article Title: GLIAL ANKYRINS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY

doi: 10.1038/nn.3858

Figure Lengend Snippet: AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155. ( a–d ) P8 sciatic nerve sections were stained for AnkB (N105/17), NFasc and Caspr ( a, b ), and the respective longitudinal line scans are shown ( c, d ). ( e ) Immunoprecipitation of AnkB from adult rat sciatic nerves co-precipitated NF155. Immunoprecipitation with the anti-GFP antibody served as a negative control. Heavy chain, mouse IgG heavy chain. AnkB was detected by H-300 rabbit polyclonal antibodies. ( f ) Immunoprecipitation of AnkG from P21 mouse spinal cords co-precipitated NF186 and NF155. AnkG was detected by the goat polyclonal antibodies. The immunoprecipitation in ( e, f ) was reproduced at least three times. The full blots are presented in . ( g–i ) Immunostaining of P7 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the residual AnkB and NFasc at paranodes. ( j–l ) Immunostaining of P7 spinal cords (AnkG, N106/36). Arrows point to the residual AnkG and NFasc at paranodes. ( m–o ) Immunostaining of P5 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the paranodes with residual AnkB. ( p–r ) Immunostaining of P12 Nfasc-cHet ( Cnp-Cre;Nfasc f/+ ) and Nfasc-cKO ( Cnp-Cre;Nfasc f/f ) spinal cords (AnkG, N106/36). Two mice per genotype and more than 100 nodes were examined in each mouse. 80–190 nodes per animal were quantified. Scale bars = ( a, b ) 5 μm for (a) and 3.3 μm for (b); 5 μm ( g–i , j–l , m–o , and p–r ).

Article Snippet: The primary antibodies used were: rabbit antibodies against AnkB membrane-binding domain , AnkB C-terminal region (H-300, SantaCruz), AnkG spectrin-binding domain and C-terminal region , AnkG exon 1b and exon 1e (generated by immunizing against peptides unique to these domains followed by affinity purification of specific antibodies), AnkG 480/270 (generated, affinity purified, and kindly provided by Dr. Paul Jenkins, Duke University), AnkR (generated against His-tagged, full-length human AnkR and affinity purified), GFP (A11122, Life Technologies), NF-M (AB1987, EMD Millipore), Caspr , βIV spectrin SD , Kv1.2 , Caspr2 (ab33994, Abcam), Nav1.2 , Nav1.6 , and 4.1G (kindly provided by Dr. Elior Peles); mouse monoclonal antibodies against AnkB membrane-binding domain (N105/13 and N105/17, UC Davis/NIH NeuroMab Facility), AnkB spectrin-binding domain (2.20, SantaCruz), AnkG spectrin-binding domain or C-terminal region (N106/36 and N106/65, NeuroMab), Caspr (K65/35, NeuroMab), contactin (K73/20, NeuroMab), actin (C4, EMD Millipore), ZO-1 (ZO1-1A12, Life Technologies), E-cadherin (4A2C7, Life Technologies), Kv1.2 (K14/16, NeuroMab), Nav1.6 (K87A/10, NeuroMab), CNP (11-5B, Sigma-Aldrich), MAG (513, EMD Millipore), MBP (SMI-94, Covance), Necl4 (N244/5, NeuroMab), pan-Nav channels (K58/35 ) and GFP (N86/38, NeuroMab); the rat monoclonal antibody against MBP (MAB386, EMD Millipore); chicken antibodies against NFasc (AF3235, R&D Systems) and βIV spectrin SD ; goat antibodies against contactin (AF904, R&D Systems), connexin 32 (C-20, Santa Cruz), and AnkG (generated against the C-terminal domain of AnkG minus the death domain and affinity purified).

Techniques: In Vivo, Staining, Immunoprecipitation, Negative Control, Immunostaining

Journal: Developmental biology

Article Title: Identification of a starfish egg PLC-gamma that regulates Ca2+ release at fertilization.

doi: 10.1016/j.ydbio.2004.01.031

Figure Lengend Snippet: Fig. 2. The full-length AmPLC-g cDNA clone encodes a protein recognized by anti-PLC-g antibodies. The full-length AmPLC-g cDNA clone was transcribed and translated in the presence of 35S-methionine. The reaction mixture (lane 1 is one-tenth of the total TNT reaction) was divided, diluted in buffer, and immunoprecipitated with 5 Ag of a mixed monoclonal antibody against bovine PLC-g1 (lane 3), affinity-purified anti-AmPLCgpep (lane 5), or anti-AmPLCgSH2 (lane 7). Protein G sepharose (lane 2), depleted AmPLCpep antibody (lane 4), and anti-chicken IgY agarose beads (lane 6) were incubated alone with the reaction mixture as negative controls. Immunoprecipitates were electrophoresed on a 7.5% polyacrylamide SDS gel. The dye front was allowed to run off for 45 min to increase the resolution of the high Mr protein. The gel was fixed and treated with EnHance, then dried and exposed to x-ray film. The arrow denotes the position of the recombinant AmPLC-g protein. Lanes 1–5 are a 30-h exposure; lanes 6 and 7 are a 96-h exposure. As another control for specificity, the antibodies were used to immunoprecipitate reaction mixtures of an unrelated protein, luciferase. None of the three anti-PLC-g antibodies recognized the control protein (data not shown). Precision size standards are 1000 Da. The experiment was repeated three times with the same results.

Article Snippet: An HRP-conjugated anti-chicken IgY secondary antibody (Aves Labs) was used at 1:15,000.

Techniques: Immunoprecipitation, Affinity Purification, Incubation, SDS-Gel, Recombinant, Control, Luciferase

Journal: Developmental biology

Article Title: Identification of a starfish egg PLC-gamma that regulates Ca2+ release at fertilization.

doi: 10.1016/j.ydbio.2004.01.031

Figure Lengend Snippet: Fig. 3. The AmPLC-g antibodies specifically recognize PLC-g protein in echinoderm eggs. Eggs from S. purpuratus (Sp; lane 1) and A. miniata (Am, lanes 2 and 3) were lysed in PLC isolation buffer (lanes 1 and 2) or directly in Laemmli sample buffer (lane 3; see Materials and methods). After clarification by centrifugation, total soluble proteins (20 Ag) were subjected to electrophoresis on 7.5% polyacrylamide gels and transferred to nitrocellulose. After blocking, blots were probed with 2 Ag/ml of the affinity-purified AmPLCpep antibody (A) or the anti-AmPLCSH2 antibody (B). Antibody binding was detected with HRP-conjugated anti-chicken IgY and enhanced chemiluminescence (a 2-min exposure is shown). The arrows indicate the intact PLC-g protein. The lower bands (arrowheads) probably are degradation products (see Results). Size standards (1000 Da) are indicated at left. The experiment (using the optimal buffer conditions) was performed on nine different batches of eggs. Shown are results typical of seven of those experiments. In the other two experiments, significant degradation of the intact PLC-g was observed and the number of immunoreactive bands recognized by the AmPLCSH2 antibody increased (see Results).

Article Snippet: An HRP-conjugated anti-chicken IgY secondary antibody (Aves Labs) was used at 1:15,000.

Techniques: Isolation, Clarification Assay, Centrifugation, Electrophoresis, Blocking Assay, Affinity Purification, Binding Assay

Journal: Developmental biology

Article Title: Identification of a starfish egg PLC-gamma that regulates Ca2+ release at fertilization.

doi: 10.1016/j.ydbio.2004.01.031

Figure Lengend Snippet: Fig. 8. Fertilization-dependent tyrosine phosphorylation of echinoderm PLC-g. Unfertilized (U) and fertilized (F; 2.5 min post sperm addition) S. purpuratus eggs were lysed in PLC isolation buffer (see Materials and methods). After clarification by centrifugation, the lysates (lanes 1 and 2; 20 Ag of total soluble lysate input) were adjusted to 3 mg/ml and incubated with 5 Ag AmPLCpep antibody and anti-chicken IgY agarose beads (lanes 4 and 6) or beads alone as a control (‘‘C’’; lanes 5 and 7). Incubation of 5 Ag AmPLCpep antibody and anti-chicken IgY agarose beads in the lysis buffer served as a mock IP control (M; lane 3). Washed IPs were split into two samples, electrophoresed on 7.5% polyacrylamide SDS gels, transferred to nitrocellulose, blocked and probed with (A) AmPLCpep antibody or (B) PY20 monoclonal antibody. Panel C shows a replicate blot probed with the PY20 monoclonal antibody in the presence of free ortho-phoshotyrosine as a competitor, demonstrating the specificity of the antibody. The experiment was repeated five times on five different batches of eggs with the same results in four of the experiments. In the fifth experiment, significant degradation of the intact PLC-g protein was observed and no tyrosine phosphorylation was detected on the remaining intact protein (data not shown). The competitor experiment was repeated twice, with the same result. Precision Mr standards are indicated (1000 Da).

Article Snippet: An HRP-conjugated anti-chicken IgY secondary antibody (Aves Labs) was used at 1:15,000.

Techniques: Phospho-proteomics, Isolation, Clarification Assay, Centrifugation, Incubation, Control, Lysis

Journal: Xenotransplantation

Article Title: Characterization of immunogenic Neu5Gc in Bioprosthetic Heart Valves

doi: 10.1111/xen.12260

Figure Lengend Snippet: Characterization of sialic acids in xenogenic cardiac tissues by ELISA. Tissue homogenates of native porcine aortic valve cusp (A), pulmonary valve cusp (B), pericardium (C), and bovine pericardium (D) were coated onto a 96-well plate and analyzed with anti-Neu5Gc IgY (Neu5Gc), biotinylated-SNA lectin or biotinylated-MAL-II lectin (detect α2–6-linked or α2–3-linked sialic-acids, respectively), washed then detected with HRP-anti-chicken IgY or HRP-streptavidin, respectively. All tissue samples express Neu5Gc, with higher levels in pericardium compared to valve cusps (at least two different samples from each tissue were tested, data represent three independent experiments; mean ± SEM).

Article Snippet: Antibodies and Lectins We used affinity-purified polyclonal chicken anti-Neu5Gc IgY [ 40 ] (Biolegend), horse radish peroxidase (HRP)-streptavidin , Cy3-streptavidin, HRP-goat-anti-human IgG, Cy3-goat-anti-human IgG (H+L), biotinylated donkey-anti-chicken IgY, HRP-AffiniPure donkey-anti-chicken IgY (IgG)(H+L), Cy3-donkey-anti-chicken IgY (IgG)(H+L) (Jackson ImmunoResearch), biotinylated GSL I – isolectin B4 (IB4), biotinylated SNA ( Sambucus nigra ) and biotinylated MAL-II ( Maackia amurensis lectin II) (Vector Labs).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Xenotransplantation

Article Title: Characterization of immunogenic Neu5Gc in Bioprosthetic Heart Valves

doi: 10.1111/xen.12260

Figure Lengend Snippet: Analysis of Sia expression in commercial BHV by microarray and by DMB-HPLC. (A) Indicated BHV homogenates (100 ng/μl) were printed at ten replicates on epoxide-coated slides and detected for Neu5Gc, α-Gal, α2–3-linked or α2–6-linked sialic acids by Cy3-anti-Neu5Gc IgY (Neu5Gc) or biotinylated-IB4/MAL-II/SNA lectins, respectively, and binding detected by Cy3-strepavidin. Intensity unit are Relative Fluorescence Units (RFU; representing fluorescence at 532 nm after local background subtraction (representative of two independent experiments; mean ± SD). All BHV showed more Neu5Gc than α-Gal and more Siaα2–3 than Siaα2–6. (B) BHV homogenates were acid hydrolyzed with sulfuric acid and analyzed by DMB-HPLC for quantifying Neu5Ac and Neu5Gc (single BHV samples of each brand; two independent experiments; mean ± SEM). Neu5Gc is clearly detected in all tested BHV.

Article Snippet: Antibodies and Lectins We used affinity-purified polyclonal chicken anti-Neu5Gc IgY [ 40 ] (Biolegend), horse radish peroxidase (HRP)-streptavidin , Cy3-streptavidin, HRP-goat-anti-human IgG, Cy3-goat-anti-human IgG (H+L), biotinylated donkey-anti-chicken IgY, HRP-AffiniPure donkey-anti-chicken IgY (IgG)(H+L), Cy3-donkey-anti-chicken IgY (IgG)(H+L) (Jackson ImmunoResearch), biotinylated GSL I – isolectin B4 (IB4), biotinylated SNA ( Sambucus nigra ) and biotinylated MAL-II ( Maackia amurensis lectin II) (Vector Labs).

Techniques: Expressing, Microarray, Binding Assay, Fluorescence