Journal: Arthritis Research & Therapy

Article Title: Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

doi: 10.1186/s13075-018-1710-0

Figure Lengend Snippet: Downregulation of mPR3 after treating neutrophils with PECAM-1. a interaction between platelet endothelial cell adhesion molecule-1 (PECAM-1) and CD177 at various concentrations. b Representative histogram of the effect of PECAM-1 on membrane-bound proteinase-3 (mPR3) expression in a dose-dependent manner. c Incubation of TNF-α-primed neutrophils with PECAM-1 at 30 μg/ml significantly decreased mPR3 expression. Bars denote means ± SD of mPR3 expression (mean fluorescence intensity; MFI). d Incubation of TNF-α-primed neutrophils with PECAM-1 at 30 μg/ml significantly increased proteinase-3 (PR3) levels in the supernatant. Bars denote means ± SD of PR3 concentration (ng/ml). Neutrophils treated with phorbol myristate acetate (PMA) were employed as positive control. * p < 0.05. FACS fluorescence-activated cell sorting, JAM-1 junctional adhesion molecule-1, OD optical density

Article Snippet: Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Incubation, Fluorescence, Concentration Assay, Positive Control, FACS

Journal: Arthritis Research & Therapy

Article Title: Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

doi: 10.1186/s13075-018-1710-0

Figure Lengend Snippet: PECAM-1 incubation decreased antineutrophil cytoplasmic antibody (ANCA)-induced respiratory burst of neutrophils. Neutrophil respiratory burst detected by DHR assay was performed after proteinase-3 (PR3)-ANCA immunoglobulin (Ig)G incubation for 1 h. Neutrophils treated with phorbol myristate acetate (PMA) were employed as positive control. Bars denote means ± SD of Rhodamine 123 expression (mean fluorescence intensity; MFI). * p < 0.05. JAM-1 junctional adhesion molecule-1, PECAM-1 platelet endothelial cell adhesion molecule-1, PR3 ANCA PR3-ANCA-positive IgGs, TNF-α tumor necrosis factor-alpha

Article Snippet: Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Incubation, Positive Control, Expressing, Fluorescence

Journal: Arthritis Research & Therapy

Article Title: Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

doi: 10.1186/s13075-018-1710-0

Figure Lengend Snippet: PECAM-1 incubation decreased antineutrophil cytoplasmic antibody (ANCA)-induced degranulation of neutrophils. Lactoferrin is considered as a biomarker of neutrophil degranulation and was detected after proteinase-3 (PR3)-ANCA immunoglobulin (Ig)G incubation for 1 h. Neutrophils treated with phorbol myristate acetate (PMA) were employed as positive control. Bars denote means ± SD of lactoferrin concentration (ng/ml). * p < 0.05. JAM-1 junctional adhesion molecule-1, PECAM-1 platelet endothelial cell adhesion molecule-1, PR3 ANCA PR3-ANCA-positive IgGs, TNF-α tumor necrosis factor-alpha

Article Snippet: Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Incubation, Biomarker Assay, Positive Control, Concentration Assay

Journal: Arthritis Research & Therapy

Article Title: Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

doi: 10.1186/s13075-018-1710-0

Figure Lengend Snippet: Effect of PECAM-1 on degranulation in CD177-negative neutrophils. a SHP-1 phosphorylation was detected in neutrophils incubated with platelet endothelial cell adhesion molecule-1 (PECAM-1). b Effect of PECAM-1 on degranulation of CD177-negative and mixed neutrophils. Degranulation induced by proteinase-3 (PR3)-antineutrophil cytoplasmic antibody (ANCA)-positive immunoglobulin (Ig)Gs (PR3 ANCA) was little influenced by preincubation with soluble PECAM-1 in CD177-negative neutrophils, but it was significantly inhibited by soluble PECAM-1 in mixed neutrophils. The level of lactoferrin was expressed as a percentage of control in each subset. The purity of the CD177-positive subset in mixed neutrophils was 72.7 ± 10.7% and the purity of CD177-negative neutrophils after selecting was 86.4 ± 8.5%. Bars represent mean ± SD of repeated measurements from four independent experiments. ** p < 0.01, *** p < 0.001. MW molecular weight, PMA phorbol myristate acetate, TNF-α tumor necrosis factor-alpha

Article Snippet: Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Incubation, Molecular Weight

Journal: Arthritis Research & Therapy

Article Title: Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

doi: 10.1186/s13075-018-1710-0

Figure Lengend Snippet: Platelet endothelial cell adhesion molecule-1 (PECAM-1) interaction with CD177 decreased GEnC activation and injury induced by patient-derived proteinase-3 (PR3)-antineutrophil cytoplasmic antibody (ANCA)-positive immunoglobulin (Ig)Gs (PR3 ANCA). By preincubation with PECAM-1, the levels of soluble intercellular cell adhesion molecule-1 (sICAM-1) significantly decreased in the supernatants of GEnCs treated with neutrophil plus patient-derived PR3-ANCA-positive IgGs. Bars denote means ± SD of sICAM-1 concentration (pg/mL). * p < 0.05. JAM-1 junctional adhesion molecule-1, PMA phorbol myristate acetate, TNF-α tumor necrosis factor-alpha

Article Snippet: Recombinant PECAM-1, junctional adhesion molecule-1 (JAM-1), and CD177 were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Activation Assay, Derivative Assay, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: The Semiconserved Head Structure of Plasmodium falciparum Erythrocyte Membrane Protein 1 Mediates Binding to Multiple Independent Host Receptors

doi:

Figure Lengend Snippet: Binding of Different Receptors and RBCs to var Fragment–transfected COS-7 Cells

Article Snippet: Soluble PECAM-1/CD31 purified from Chinese hamster ovary (CHO) cells (cat. no. ADP6) was purchased from R&D Systems.

Techniques: Binding Assay, Transfection

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 2. Cytotoxicity, Oligomer Formation, and Lethal Effects of CPB Depend on CD31 Expression (A) Viability of bEndwt, bEndCD31ko, and bEnd- CD31ko transduced with CD31-GFP or CD54-GFP incubated with indicated doses of CPB (24 h, 37C) as a percentage of untreated control cells. See also Figure S2A. (B) Viability of WT EpH4 and EpH4 cells stably expressing CD31-GFP or CD54-GFP incubated as in (A). Data in (A and B) are represented as means (N = 8) ± SD. Multiple comparison two-way ANOVA, Sidak’s multiple comparison test, values with p < 0.0001 are indicated by an asterisk. (C) Western blots of EpH4, bEndwt, and bEnd- CD31ko incubated with 8 mg/mL CPB for 30 min at 37C and probed with indicated antibodies. (D) Effect of CD31 depletion on the lethal activity of CPB in C57BL/6 mice. WT and CD31-knockout mice were injected (i.p.) with 1 mg CPB per 20 g body weight. As a control, WT mice were injected with neutralized CPB. WT mice (n = 16), CD31- knockout mice (n = 17), and control (n = 13). Log- rank test, * significance (p < 0.0001). See also Figure S3A.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Expressing, Transduction, Incubation, Control, Stable Transfection, Comparison, Western Blot, Activity Assay, Knock-Out, Injection

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 3. CPB Targets CD31 to Kill Endothelial Cells (A) Immunofluorescence micrographs of bEndwt cells incubated in the presence and absence of 1 mg/mL CPB (10 min, 37C). CPB (green), CD31 (red), DNA (blue). Scale bar, 30 mm. (B) Left panel: maximum intensity projections of z stacks of representative micrographs of in situ PLA of bEndwt and bEndCD31ko cells incubated in the presence and absence of 1 mg/mL CPB (10 min, 37C). PLA signal (red), DNA (blue). Scale bar, 60 mm. Right panel: Box plots of quantification of PLA puncta per cell type. ANOVA results, ns (non-significant), ***3 (p < 0.0001), N = 30. (C) Immunoblots of co-immunoprecipitation experiments of CD31-GFP expressing cells incubated with 4 mg/mL CPB and lysed (1.5% digitonin). CD31-GFP was immunoprecipitated with anti-GFP beads, anti-myc beads were used as control. Immunoblots containing 5% input (In) and 50% eluate (elu) were probed with anti-GFP and anti-CPB antibodies. E-cadherin and VE-cadherin served as a control. See also Figures S3B–S3D.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Incubation, In Situ, Western Blot, Immunoprecipitation, Expressing, Control

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 4. CD31 Extracellular Ig6 Domain Is Required for CPB Targeting of Endothelial Cells (A) Top: schematic protein domain structures of CD31 with N-terminal signal peptide (SP), the extracellular domain (ED) containing the six Ig-like domains (Ig1– Ig6), the single transmembrane helix (TM), and the intracellular domain (ID). Below, corresponding exons. Constructs below show all different GFP fusion proteins used. Deleted exons are indicated as D in the protein name. Constructs that sensitized resistant cells are indicated in black, constructs that were non-sensitizing in gray. *Construct CD31D(3–7,10–16) did not localize at the plasma membrane and was not investigated further. (B and C) Viability of cell lines expressing indicated constructs after incubation with 1 mg/mL CPB (24 h, 37C) in % to untreated control cells. Data are represented as means (N = 8) ± SD. Two-way ANOVA, Sidak’s multiple comparison test, ***3 (p < 0.0001). (D) Co-immunoprecipitation experiment of bEndCD31ko cell lines expressing different CD31-GFP truncation mutants incubated with 4 mg/mL CPB for (20 min, 37C). IP: anti-GFP beads. Immunoblots containing 5% input (In) and 50% eluate (elu) and were probed with anti-GFP and CPB antibodies. The two upper blots show the same membrane probed with differently labeled fluorescent secondary antibodies. VE-cadherin served as a control on the same blot. See also Figures S4–S6.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Construct, Clinical Proteomics, Membrane, Expressing, Incubation, Control, Comparison, Immunoprecipitation, Western Blot, Labeling

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 5. Highly Conserved Region in Ig6 Is Required for CPB Cytotoxicity (A) Sequence alignments of the CD31 Ig6 domains of mus musculus, homo sapiens, and sus scrofa. Alignments were produced using EMBL-EBI server (https:// www.ebi.ac.uk/Tools/msa/clustalo). Identical residues are indicated by dark gray boxes, residues that belong to the same amino acid group by light gray boxes. Mutations are indicated with gray brackets if they were inconsequential or black if mutation diminished sensitivity to CPB. Black arrows indicate beta sheets, the dotted line indicates disulfide bond. (B) Viability of transduced HEK 293FT cells expressing five mouse CD31 Ig6 mutants (alanine and valine substitutions) after incubation with CPB (1 mg/mL, 24 h, at 37C) in % to untreated control cells. Data are represented as means (N = 12) ± SD. Two-way ANOVA, Sidak’s multiple comparison test, ***3 (p < 0.0001). (C) Schematic protein domain structures of species chimeras of mouse CD31 with either substituted pig or human Ig6 domain. (D) Viability of transduced HEK 293FT cells expressing indicated cimeras from C, mut indicates an alanine valine mutation at position 527–536 (mouse) or 537–546 (human and porcine). Data are represented as means (N = 16) ± SD. Two-way ANOVA, Sidak’s multiple comparison test, ***3 (p < 0.0001). See also Figure S6.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Sequencing, Produced, Mutagenesis, Expressing, Incubation, Control, Comparison

Journal: Cell host & microbe

Article Title: CD31 (PECAM-1) Serves as the Endothelial Cell-Specific Receptor of Clostridium perfringens β-Toxin.

doi: 10.1016/j.chom.2020.05.003

Figure Lengend Snippet: Figure 6. CPB Induces Rapid Content Leakage from CD31 Containing Liposomes (A) SDS PAGE (silver stained) and western blots showing purified mouse CD31 and CD31D8 (elu) from transduced HEK293T whole cell extracts (wcl). (B) Top, schematic illustrating the liposome floatation assay. Liposomes containing CD31 or CD31D8, or empty liposomes were incubated for 2 h with 10 mg/mL CPB and floated through a sucrose gradient by centrifugation. A sample before gradient centrifugation (crude) and the three fractions (top, middle, bottom) were analyzed by SDS-PAGE and western blotting. (C) Kinetic traces from carboxyfluorescein leakage assay of CD31-LUVs, CD31D8-LUVs, and empty LUVs treated with indicated dose of CPB or buffer as control (CD31, buffer)Arrowheads indicate addition of toxins or buffer after 20 s. Fluorescence was normalized to maximal fluorescence after complete solubilization of LUVs with detergent. (D) Kinetic traces from carboxyfluorescein leakage assay of CD31D8-LUVs and empty LUVs treated with indicated PFTs.

Article Snippet: CD31susIg6 (FYREKEGKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31susIg6 pLenti as template and mutagenic primers Fw24 and Rv24 with Ta of 60 C and elongation time of 8.5 min. CD31homoIg6 was generated using Gibson cloning kit and CD31 containing pLenti plasmid that was linearized with primers Fw25 and Rv25 with Ta of 63 C and elongation time of 8.5 min. PCR product containing the Ig6 of human CD31 was generated from human CD31 (Origene) containing plasmid and primers Fw26 and Rv26, Ta of 68 C and amplification time 10 sec. CD31homoIg6 (FYKEKENKPF to AAVAAVAAVA) was generated using Q5 site-directed mutagenesis kit, CD31homoIg6 pLenti as template and mutagenic primers Fw27 and Rv27 with Ta of 59 C and elongation time of 8.5 min.

Techniques: Liposomes, SDS Page, Staining, Western Blot, Incubation, Gradient Centrifugation, Control, Fluorescence

Journal: Nature

Article Title: Capillary cell-type specialization in the alveolus.

doi: 10.1038/s41586-020-2822-7

Figure Lengend Snippet: Fig. 1 | Two stable, intermingled alveolar capillary cell types. a, Alveolar capillaries in adult mouse lung immunostained for PECAM1. b, t-distributed stochastic neighbour embedding (t-SNE) plot of endothelial cell populations annotated in scRNA-seq data for adult mouse lung13. c, Heat map of expression of capillary subset markers and the general endothelial marker Cldn5 in individual capillary cells. CPM, counts per million. d–f, Single-molecule fluorescent in situ hybridization (smFISH) for the capillary subset markers Apln (d, f) or Ednrb and Car4 (aCap) (e), and Aplnr (gCap) (d–f), in adult mouse lung. Images in d (right) and f show individual aCap and gCap cells. g, Relative abundance of aCap cells, gCap cells and cells that co-express aCap and gCap markers (intermediate (IM) cells) in lungs from 3-month-old (young) and 24-month-old (aged) mice (data shown as mean; n = 500 cells scored per mouse; 2 mice per group). h, i, Co-expression of tdTomato lineage label (asterisks) and aCap marker Ednrb but not gCap marker Aplnr (h), or gCap marker Aplnr but not aCap marker Apln (i), in lungs collected six months after mature aCap (h) or gCap (i) cells were lineage-labelled. Blue, DAPI. Scale bars, 10 μm.

Article Snippet: CD34 (BD Biosciences, 347660):https://www.bdbiosciences.com/eu/applications/research/clinical-research/oncology-research/ 3 nature research | reporting sum m ary O ctober 2018 blood-cell-disorders/surface-markers/human/purified-mouse-anti-human-cd34-my10/p/347660 Endomucin (Invitrogen, eBioV.7C7, 14-5851-82): https://www.thermofisher.com/antibody/product/Endomucin-Antibody-cloneeBioV-7C7-V-7C7-Monoclonal/14-5851-82 Integrin alpha8 (R&D, AF4076): https://www.rndsystems.com/products/mouse-rat-integrin-alpha8-antibody_af4076 Pecam1 (rat anti-mouse; BD Biosciences, 553370): https://www.bdbiosciences.com/ds/pm/tds/553370.pdf Pecam1 (mouse anti-human; R&D, BBA7): https://www.rndsystems.com/products/human-cd31-pecam-1-antibody-9g11_bba7 tdTomato (Rockland, 600-401-379): https://rockland-inc.com/store/Antibodies-to-GFP-and-Antibodies-to-RFP-600-401-379O4L_24299.aspx VE-Cadherin (R&D, AF938): https://www.rndsystems.com/products/human-ve-cadherin-antibody_af938 The following primary antibodies were used to stain turtle and/or alligator tissue.

Techniques: Expressing, Marker, In Situ Hybridization

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *

doi: 10.1074/jbc.M114.563585

Figure Lengend Snippet: Sialylated oligosaccharides inhibit the homophilic mouse PECAM interaction. A, PECAM-His and PECAM-Fc pulled down by Dynabeads protein G in the presence of a series of oligosaccharides (2-(trimethylsilyl)ethyl (SE) glycoside forms, 0, 0.2, 1, and 5 mm) were evaluated by Western blot analysis. WB, Western blot. B, the relative levels of PECAM-His bound to immobilized PECAM-Fc in the presence of a series of oligosaccharides (0.2, 1, and 5 mm) were quantified by Western blot analysis. The data are shown as the means ± S.E. when the level of PECAM-His in the absence of an oligosaccharide was set at 100% (n = 3).

Article Snippet: The sources of the materials used in this study were as follows: tissue culture media and reagents, including DMEM and Dynabeads protein G from Invitrogen, Arthrobacter ureafaciens sialidase from Nacalai Tesque, Vibrio cholera sialidase and recombinant peptide N -glycosidase F from New England Biolabs, recombinant mouse PECAM from R&D Systems, human PECAM-His from Reprokine, nickel-Sepharose and Sephadex G-10 from GE Healthcare, protein molecular weight standards from Bio-Rad, and all other chemicals from Sigma or Wako Chemicals.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *

doi: 10.1074/jbc.M114.563585

Figure Lengend Snippet: α2,6-Sialylated glycocluster probe binds to immobilized PECAM and endothelial cells. A, schematic structure of the sialylated glycocluster probes used in this study. The α2,3-type disialylated, α2,6-type disialylated, or asialo-type N-glycan clusters were labeled with nitrobenzoxadiazole (star) (30). B, human asialo-PECAM-His immobilized to nickel-agarose beads was incubated with the three glycocluster probes. After a brief wash the beads were observed by fluorescence microscopy. Scale bar, 200 μm. C, prefixed HUVECs were incubated with the series of glycocluster probes and observed by fluorescence microscopy. Arrowheads indicate cell borders. Scale bar, 20 μm.

Article Snippet: The sources of the materials used in this study were as follows: tissue culture media and reagents, including DMEM and Dynabeads protein G from Invitrogen, Arthrobacter ureafaciens sialidase from Nacalai Tesque, Vibrio cholera sialidase and recombinant peptide N -glycosidase F from New England Biolabs, recombinant mouse PECAM from R&D Systems, human PECAM-His from Reprokine, nickel-Sepharose and Sephadex G-10 from GE Healthcare, protein molecular weight standards from Bio-Rad, and all other chemicals from Sigma or Wako Chemicals.

Techniques: Labeling, Incubation, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *

doi: 10.1074/jbc.M114.563585

Figure Lengend Snippet: Mouse PECAM mainly possesses sialylated biantennary N-glycans. A, a series of mutant Fc-PECAMs in which each potential N-glycosylation site was mutated were purified from overexpressing COS cells, subjected to SDS-PAGE, and visualized by silver-staining. B, immunopurified PECAM (arrowhead) from mouse lung tissues was verified by SDS-PAGE and silver staining. The de-N-glycosylated anti-PECAM IgG used for immunoaffinity chromatography is also shown (right lane). The only contaminant except for IgG, shown by the asterisk, was shown not to have any N-glycans because it was resistant to peptide N-glycosidase F treatment. C, PA-labeled N-glycans released from PECAM were separated by anion-exchange HPLC. The numbers shown indicate the eluted positions of standard N-glycans having 0, 1, 2, 3, and 4 sialic acid residues, respectively.

Article Snippet: The sources of the materials used in this study were as follows: tissue culture media and reagents, including DMEM and Dynabeads protein G from Invitrogen, Arthrobacter ureafaciens sialidase from Nacalai Tesque, Vibrio cholera sialidase and recombinant peptide N -glycosidase F from New England Biolabs, recombinant mouse PECAM from R&D Systems, human PECAM-His from Reprokine, nickel-Sepharose and Sephadex G-10 from GE Healthcare, protein molecular weight standards from Bio-Rad, and all other chemicals from Sigma or Wako Chemicals.

Techniques: Mutagenesis, Purification, SDS Page, Silver Staining, Chromatography, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *

doi: 10.1074/jbc.M114.563585

Figure Lengend Snippet: Sialidase or α2,6-sialylated oligosaccharide treatment causes PECAM internalization in endothelial cells. A, HUVECs were treated with V. cholera sialidase, fixed, and stained with anti-early endosome antigen 1 (EEA1; green) and PECAM (red) antibodies and DAPI (blue). B, HUVECs were incubated with 2 mm lactose, α2,3- or α2,6-sialylated lactose, or α2,6-sialylated or asialo-biantennary N-glycan for 18 h, fixed, and stained with PECAM (red) antibody. Scale bar, 20 μm.

Article Snippet: The sources of the materials used in this study were as follows: tissue culture media and reagents, including DMEM and Dynabeads protein G from Invitrogen, Arthrobacter ureafaciens sialidase from Nacalai Tesque, Vibrio cholera sialidase and recombinant peptide N -glycosidase F from New England Biolabs, recombinant mouse PECAM from R&D Systems, human PECAM-His from Reprokine, nickel-Sepharose and Sephadex G-10 from GE Healthcare, protein molecular weight standards from Bio-Rad, and all other chemicals from Sigma or Wako Chemicals.

Techniques: Staining, Incubation