lca conjugated agarose beads  (Vector Laboratories)

Journal: Journal of Cancer

Article Title: Identification of LCA-binding Glycans as a Novel Biomarker for Esophageal Cancer Metastasis using a Lectin Array-based Strategy

doi: 10.7150/jca.43806

Figure Lengend Snippet: Effects of LCA on EC cell migration and invasion. (A) Representative images and the mean migration number for each treatment group. (B) Representative images and the mean invasion number for each treatment group. (C) Cell viability for each treatment group. (D) Expression levels of the MMPs and PI3K/Akt pathway-associated proteins for cells treated with LCA at the IC50 concentration. Untreated cells served as a control. * P < 0.05 compared to the control group.

Article Snippet: To capture the glycoproteins, proteins from whole-cell extractions were incubated with LCA-conjugated agarose beads (Vector Labs) at 4°C overnight.

Techniques: Migration, Expressing, Concentration Assay

Journal: Journal of Cancer

Article Title: Identification of LCA-binding Glycans as a Novel Biomarker for Esophageal Cancer Metastasis using a Lectin Array-based Strategy

doi: 10.7150/jca.43806

Figure Lengend Snippet: Effects of tunicamycin on EC cell migration and invasion. (A) Cell viability for each treatment group. (B) Representative images and the mean migration number for cells treated with tunicamycin at the IC50 concentration. (C) Representative images and the mean invasion number for cells treated with tunicamycin at the IC50 concentration. (D) Expression levels of LCA-binding glycans for cells treated with tunicamycin at the IC50 concentration. (E) Expression levels of the MMPs and PI3K/Akt pathway-associated proteins for cells treated with tunicamycin at the IC50 concentration. Cells treated with DMSO served as a control. * P < 0.05 compared to the control group.

Article Snippet: To capture the glycoproteins, proteins from whole-cell extractions were incubated with LCA-conjugated agarose beads (Vector Labs) at 4°C overnight.

Techniques: Migration, Concentration Assay, Expressing, Binding Assay

Journal: Journal of Cancer

Article Title: Identification of LCA-binding Glycans as a Novel Biomarker for Esophageal Cancer Metastasis using a Lectin Array-based Strategy

doi: 10.7150/jca.43806

Figure Lengend Snippet: Identification of CD147 as an LCA-recognized glycoprotein. (A) Coomassie blue staining for immunoprecipitated samples. (B) LCA immunoprecipitation assay for Eca-109 and KYSE-150 cells. PD: pull down. (C) Representative photomicrographs for CD147 expression in EC and adjacent non-tumor tissues. Scale bar represents 100 µm. (D) Analysis of CD147 expression based on the scoring data for IHC (E) Correlation between LCA-binding glycans and CD147 expression in EC tissues. IgG served as a control. P < 0.05.

Article Snippet: To capture the glycoproteins, proteins from whole-cell extractions were incubated with LCA-conjugated agarose beads (Vector Labs) at 4°C overnight.

Techniques: Staining, Immunoprecipitation, Expressing, Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness

doi: 10.1186/s13058-022-01513-3

Figure Lengend Snippet: FUT8 modulates core fucosylation of IL6ST protein and its downstream signaling. a and b IL6ST and OSM receptor (OSMR) proteins core fucosylated by FUT8 in HEK-293 T cells. Recombinant IL6ST or OSMR proteins in control or two FUT8-KO 293 T cell lines were probed with biotinylated LCA, then detected with streptavidin-conjugated horseradish peroxidase. Protein domain organization of these selected proteins is illustrated according to Uniprot data ( http://uniprot.org ). Amino acid number and potential N -linked glycosylation sites are marked on the upper and lower side, respectively. Core-fucosylated sites confirmed by LC–MS/MS analyses are in red. TM, transmembrane domain. c Core-fucosylation sites of IL6ST protein. Shows LC–MS/MS summed extracted ion chromatogram (XIC) of identified core-fucosylated glycopeptides for IL6ST. The number on asparagine (N) indicates the position in the protein sequence. d FUT8 KO impaired IL-6 and OSM signaling in STAT3 reporter HEK cells. Parental or FUT8-KO HEK-Blue™ IL-6 cells (InvivoGen) were treated with recombinant IL-6 (upper) or OSM (lower). Levels of STAT3-inducible secreted embryonic alkaline phosphatase (SEAP) indicating STAT3 activity were monitored by using QUANTI-Blue. Data are mean ± SD. ** p < 0.01, * p < 0.05, FUT8-KO #1 or FUT8-KO#2 versus parental HEK-Blue™ IL-6 cells. e FUT8 KO impaired IL-6 and OSM signaling in MDA-MB-231 cells. Control and FUT8-KO MDA-MB-231 cells were treated with the indicated concentrations of recombinant IL-6 (left panel) or OSM (right panel) for 15 min. Cell lysates underwent western blot analysis with antibody for phosphorylated STAT3 or total STAT3

Article Snippet: The core fucosylated glycoproteins were enriched with LCA-agarose beads (Vector Laboratories).

Techniques: Recombinant, Liquid Chromatography with Mass Spectroscopy, Sequencing, Activity Assay, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness

doi: 10.1186/s13058-022-01513-3

Figure Lengend Snippet: FUT8 modulates core fucosylation and adhesive capability of αvβ5 integrins. a Integrins αv and β5 were core fucosylated by FUT8 in HEK-293 T cells. Recombinant integrin αv (upper panel) and integrin β5 (lower panel) in the control or two FUT8-KO 293 T cell lines were probed with biotinylated LCA, then detected with streptavidin-conjugated horseradish peroxidase. Protein domain organization of these selected proteins are depicted according to Uniprot data. Amino acid number and potential N-linked glycosites are marked on the upper and lower side, respectively. Core-fucosylated sites verified by LC–MS/MS analysis are in red. TM, transmembrane domain. b Core fucosylation of integrin αv. LC–MS/MS summed extracted ion chromatogram (XIC) of identified core-fucosylated glycopeptides for integrin αv. The number on asparagine (N) indicates the position in the protein sequence. c and d Functional blocking antibodies abolished cell adhesion to vitronectin and laminin-5 in MDA-MB-231 cells. Plates were pre-coated with 5 μg/ml vitronectin ( c ) or laminin-5 ( d ). MDA-MB-231 cells were pre-incubated with anti-integrin antibodies (10 μg/ml) before cells were placed on coated plates for 10 min at 37 °C. Adherent cells were quantified after fixation and stained with crystal violet. Data are mean ± SD. ** p < 0.01 compare with control IgG. e and f FUT8 KO reduced cell adhesion to vitronectin and laminin-5 in MDA-MB-231 cells. Parental or FUT8-KO MDA-MB-231 cells were allowed to attach to vitronectn-coated ( e ) or laminin-5-coated ( f ) plates. Adherent cells were quantified after fixation and stained with crystal violet. Data are mean ± SD. ** p < 0.01, FUT8-KO versus parental cells

Article Snippet: The core fucosylated glycoproteins were enriched with LCA-agarose beads (Vector Laboratories).

Techniques: Recombinant, Liquid Chromatography with Mass Spectroscopy, Sequencing, Functional Assay, Blocking Assay, Incubation, Staining

Journal: Breast Cancer Research : BCR

Article Title: Quantitative glycoproteomics analysis identifies novel FUT8 targets and signaling networks critical for breast cancer cell invasiveness

doi: 10.1186/s13058-022-01513-3

Figure Lengend Snippet: Validation of the SILAC results of identified glycoproteins. Core fucosylation of the selected proteins was eliminated by FUT8 knockout (KO). Recombinant proteins in the control or two FUT8-KO MDA-MB-231 cell lines were probed with biotinylated LCA, then detected with streptavidin-conjugated horseradish peroxidase. Protein domain organization of these selected proteins was depicted according to the Uniprot database ( https://www.uniprot.org ); potential N-linked glycosylation sites are marked. TM transmembrane domain

Article Snippet: The core fucosylated glycoproteins were enriched with LCA-agarose beads (Vector Laboratories).

Techniques: Knock-Out, Recombinant