biotinylated leucoagglutinin  (Vector Laboratories)

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-424 Predicts a Role for β-1,4 Branched Glycosylation in Cell Cycle Progression *

doi: 10.1074/jbc.M115.672220

Figure Lengend Snippet: Loss of MGAT4A activity does not affect GlcNAc-β-1,6 branching. A , biosynthetic pathway of tetraantennary branched N -glycans. Binding sites of PHA-L are indicated. B , analysis of PHA-L binding to the Consortium of Functional Glycomics ( CFG ) glycan array v. 5.0 indicates a preference for GlcNAc-β-1,6 branched products. The graph shows the data for 100 μg/ml biotinylated-PHA-L (Vector Laboratories) binding to glycans bearing only β-1,4 branching (β -(1,4) ) and the corresponding β-1,6 glycans (β -(1,6) ). Data are given as fluorescence intensity. C , knockdown of MGAT4A in MCF-7 cells shows no significant change in MGAT4B or MGAT5 mRNA levels by qRT-PCR. The graph shows -fold change mRNA expression compared with NTC. n = 3 biological replicates. Error bars represent the S.D. D , fluorescence microscopy of PHA-L stained MGAT4A-KD and NTC cells. No significant loss of PHA-L binding were observed. Images shown are representative of two biological replicates, five images per replicate.

Article Snippet: Samples were then blocked with 5% bovine serum albumin in HBSS (2 ml, 30 min) and incubated with primary antibodies or lectins diluted in HBSS as follows for 1 h at room temperature: α-galectin-3 (1:250), α-E-cadherin (1:500), biotinylated-PHA-L (1:500, Vector Laboratories).

Techniques: Activity Assay, Binding Assay, Functional Assay, Plasmid Preparation, Fluorescence, Quantitative RT-PCR, Expressing, Microscopy, Staining

Journal: Nature Communications

Article Title: Programmed cell removal by calreticulin in tissue homeostasis and cancer

doi: 10.1038/s41467-018-05211-7

Figure Lengend Snippet: Cell surface asialoglycans regulates CRT-mediated PrCR. a , b Treatment with neuraminidase led to the removal of sialic acids from the cell surface of HL60 cells. HL60 cells were treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Cell surface sialic acids were examined by staining with EBL (a) and MAL (b) by flow cytometry analysis. EBL, Elderberry Bark Lectin; MAL, Maackia Amurensis Lectin II. c , d Examination of cell surface CRT and PHA-L binding sites on cancer cells. HL60 cells were treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Recombinant CRT ( c ) and PHA-L ( d ) binding after treatment were measured by flow cytometry. e , f Phagocytosis of cancer cells with neuraminidase treatment. In vitro Phagocytosis assays were performed with HL60, K562, DLD-1, and SW620 cells treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu) as target cells. Mouse bone marrow-derived ( e ) and human peripheral blood monocyte-derived ( f ) macrophages were used for the assay. Phagocytosis was normalized to the maximal response in the experiments. n = 3. * P

Article Snippet: For CRT-binding experiments, cells were incubated with recombinant calreticulin human Fc fusion protein at 40 µg ml−1 or biotin-PHA-L 6 µg ml− 1 (B-1115, Vector laboratory) for 1 h incubated on ice.

Techniques: Staining, Flow Cytometry, Cytometry, Binding Assay, Recombinant, In Vitro, Derivative Assay

Journal: Nature Communications

Article Title: Programmed cell removal by calreticulin in tissue homeostasis and cancer

doi: 10.1038/s41467-018-05211-7

Figure Lengend Snippet: Identification of CRT-binding ligands on aged and malignant cells. a Screening for CRT-binding glycans with carbohydrate microarray. Purified CRT-IgG-Fc proteins were used to probe a carbohydrate microarray containing a number of different types of glycans, including N-, O- and sulfated-glycans. Anti-IgG-Fc antibody was used for detecting binding of CRT to glycans. Glyco-antigens were used at 0.05 and 0.25 μg μl −1 (left and right bars for each glycan). n = 3. Error bars represent standard deviation. b PHA-L binding to peritoneal neutrophils and macrophages at 0, 4, 6, 8, 24, and 72 h after thioglycollate injection in MRP8-Bcl2 mice. c , d Examination of cell surface CRT-binding sites on neutrophils. Bone marrow ( c ) or peritoneal neutrophils ( d ) were collected and treated with heat inactivated neuraminidase (Δneu) or neuraminidase (neu). Cells were incubated with PBS (control; black) or recombinant CRT proteins (red) and binding of CRT was then measured by flow cytometry with PE-conjugated anti-CRT antibody. rCRT binds to mature peritoneal neutrophils but not the immature bone marrow neutrophils. Treatment with neuraminidase led to the release of CRT-binding sites. e , f Immunofluorescent staining of CRT in HL60 ( e ) and SW620 ( f ) cells. CRT localized to perinuclear regions, vesicles, and cell surface. CRT was either expressed at a low level ( e ) or limited to the perinuclear regions ( f ), while a significant portion of PHA-L staining were observed on the cell surface ( e and f ).In a – d , MFI, mean fluorescence intensity

Article Snippet: For CRT-binding experiments, cells were incubated with recombinant calreticulin human Fc fusion protein at 40 µg ml−1 or biotin-PHA-L 6 µg ml− 1 (B-1115, Vector laboratory) for 1 h incubated on ice.

Techniques: Binding Assay, Microarray, Purification, Standard Deviation, Injection, Mouse Assay, Incubation, Recombinant, Flow Cytometry, Cytometry, Staining, Fluorescence

Journal: Oncotarget

Article Title: Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma

doi: 10.18632/oncotarget.27850

Figure Lengend Snippet: Evaluation of SLe x expression under downmodulation of N- and core 2 O-GalNAc glycans in the high-grade glioma cell line LN229 by flow cytometry. ( A ) ConA binding and ( B ) expression of SLe x after treatment with 150 nM TM for 24 h. ( C ) PHA-L binding and ( D ) expression of SLe x after treatment with 100 nM SW for 24 h. ( E ) PHA-L binding, ( F ) MGAT5 transcripts levels and ( G ) expression of SLe x after 48 h of MGAT5 or control siRNA transfection. ( H ) C2GNT1 transcripts levels and ( I ) expression of SLe x after 48 h of C2GNT1 or control siRNA transfection. Data represent means ± S.D. of three independent experiments. (A, C, E, F, H and I) T Test. (B, D, G) Mann–Whitney. * p

Article Snippet: Cells were washed with phosphate buffered saline (PBS) and incubated with Polyclonal Goat Anti-Mouse Phycoerythrin (PE) labeled anti-mouse immunoglobulins (Dako, USA) on ice for 30 min. For lectins binding, biotinylated ConA or PHA-L (Vector Laboratories, USA) were incubated with fluorescein-streptavidin (Vector Laboratories, USA) in 1% Bovine Serum-Albumin (BSA) (Sigma, USA) in PBS on ice for 30 min. Then, cells were incubated with the lectin-streptavidin complex on ice for 30 min. For mAbs or lectins, acquisition was carried out by a FACSCalibur flow cytometer (Becton Dickinson, USA) and analyzed by the FlowJo® software.

Techniques: Expressing, Flow Cytometry, Binding Assay, Transfection, MANN-WHITNEY

Journal: Oncotarget

Article Title: Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma

doi: 10.18632/oncotarget.27850

Figure Lengend Snippet: Glycophenotype characterization in high- and low-grade glioma cell lines. ( A ) SLe x expression of high- and low-grade glioma cell lines evaluated by flow cytometry. Doted lines represent limits between low, medium and high expression. High expression was considered greater than 1.5 rMFI, medium between 1.25 and 1.5 rMFI, and low as lower than 1.25 rMFI. Data represent means ± S.D (rMFI) of three independent experiments. ( B ) Representative experiment for Sle x expression. ( C ) Comparison of cell binding to lectins ConA and PHA-L by flow cytometry. ConA vs PHA-L: p

Article Snippet: Cells were washed with phosphate buffered saline (PBS) and incubated with Polyclonal Goat Anti-Mouse Phycoerythrin (PE) labeled anti-mouse immunoglobulins (Dako, USA) on ice for 30 min. For lectins binding, biotinylated ConA or PHA-L (Vector Laboratories, USA) were incubated with fluorescein-streptavidin (Vector Laboratories, USA) in 1% Bovine Serum-Albumin (BSA) (Sigma, USA) in PBS on ice for 30 min. Then, cells were incubated with the lectin-streptavidin complex on ice for 30 min. For mAbs or lectins, acquisition was carried out by a FACSCalibur flow cytometer (Becton Dickinson, USA) and analyzed by the FlowJo® software.

Techniques: Expressing, Flow Cytometry, Binding Assay

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: Electron micrographs of a PHA-L+ type 1 terminal (T1t) making synaptic contact (arrowhead) with the base of a proximal dendrite (den) emerging from an unlabeled putative pyramidal cell perikaryon (Pyr, pk). Scale bar: 0.5 µm.

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques:

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: PHA-L injections into the basal forebrain mainly label axons in the basolateral amygdalar nucleus. (A) Representative dual injections of PHA-L into the caudal basal forebrain (BF) that involved the ventral pallidum (VP) and substantia innominata (SI)

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques:

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: PHA-L labeled axons in the basolateral nucleus. (A) Photomontage of PHA-L-labeled type 1 axons (black) innervating PV+ interneurons (brown) in the BLa in a field dominated by these axons. Varicosities of type 1 axons are often large and clustered, and

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques: Labeling

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: Triple immunofluorescence labeling for PHA-L, VGAT, and neuronal markers in the BLa. PHA-L+ axons are red, VGAT is green, and neuronal markers (CB in A–E; CaMK in F) are blue. Yellow indicates colocalization of PHA-L and VGAT (i.e. GABAergic axons).

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques: Immunofluorescence, Labeling

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: Synaptic contacts from type 1 PHA-L+ terminals with the perikarya of a PV-immunoreactive interneuron and an unlabeled putative pyramidal neuron (inset in B). (A, B) Schematic drawings, at two levels, of a single parvalbumin-immunoreactive interneuronal

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques:

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: Synaptic contacts from type 1 PHA-L+ terminals onto the proximal dendrites of a PV+ interneuron (inset in B). (A) The proximal end of the lower dendrite of the PV+ interneuron (PVd) receives synaptic input (arrow) from a large PHA-L+ terminal (t1: terminal

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques:

Journal: Neuroscience

Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

doi: 10.1016/j.neuroscience.2011.03.027

Figure Lengend Snippet: (A, B) Electron micrographs of PHA-L+ terminals 2 and 3 (t2, t3) shown in , making synaptic contacts (arrows) onto the PV + interneuron (PVpk) and putative pyramidal neuron (PNpk). Scale bar: 0.5 µm.

Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

Techniques: