Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins

doi: 10.3390/molecules22071104

Figure Lengend Snippet: Detection of ConA and GNA binding to glycoproteins in S, R and T cells passaged three times in the absence or presence of either tunicamycin (0.1 μmol·dm −3 ) or GalNAc-α- O -benzyl (0.1 mmol·dm −3 ). Panel ( a ): Representative sample of flow cytometry measurement of FITC-ConA binding to S (upper plot), R (middle plot) and T (lower plot) cells. Black histogram – unlabeled S, R and T cells, red histogram – FITC-ConA-labeled S cells, blue histogram FITC-ConA-labeled R cells, and green histogram – FITC-ConA-labeled T cells. Parameter Δ (represents the difference between medians of labeled and unlabeled cells) was ascertained using this measurement. Similar measurements were obtained for S, R and T cells untreated and treated with tunicamycin and GalNAc-α- O -benzyl and documented in panels ( b ) (for ConA) and ( c ) (for GNA). Binding of lectin to untreated S cells was arbitrarily selected as 100%. White column—untreated cells; gray—column cell treated with GalNAc-α- O -benzyl and black column—cell treated with tunicamycin. Significance: * and +—value significantly differs from the corresponding value for S cells at p < 0.02 and p < 0.05, respectively. The data represent the means ± S.E.M. of five independent measurements. Panels ( d ) (for ConA) and ( e ) (for GNA) represent Eastern blot identification of glycoproteins in crude membrane fractions isolated from S, R and T cells untreated C, or treated with inhibitor of O-glycosylation (GalNAc-α- O -benzyl, O) or N-glycosylation (tunicamycin, N). Data are representative of three independent measurements. Red arrows indicate the P-gp form glycosylated with saccharides that are GNA ligands.

Article Snippet: For glycoprotein detection by Eastern blots, ConA and GNA conjugated with biotin (EY Laboratories Inc., San Mateo, CA, USA), and avidin conjugated with horseradish peroxidase (Sigma-Aldrich, San Diego, CA, USA) was used.

Techniques: Binding Assay, Flow Cytometry, Labeling, Isolation

Journal: bioRxiv

Article Title: N -glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase

doi: 10.1101/2021.12.13.472379

Figure Lengend Snippet: A) TconTS1 and D-TconTS1 were analyzed by SDS-PAGE with subsequent coomassie staining (upper panel), by Western blot analysis using an anti- Strep -tag antibody (middle panel) and by lectin blotting using ConA (lower panel). Results indicate the presence of high-mannose type N -glycans. B) MALDI-TOF MS analyses were performed to identify N -glycosylation sites of TconTS1. Glycopeptides from protease-digested TconTS1 were ConA-purified to concentrate glycopeptides and reduce the spectrum complexity. Peak lists were extracted from MALDI-TOF mass spectra, plotted with python and annotated with corresponding masses and glycopeptide fragments, respectively. Monosaccharide symbols follow the Symbol Nomenclature for Glycans (SNFG) .

Article Snippet: For ConA lectin blots, high-mannose N -glycosylated proteins were detected employing ConA-biotin (Galab, Hamburg, Germany) and the VECTASTAIN® ABC-HRP Kit (Vector Laboratories, Burlingame, CA, United States).

Techniques: SDS Page, Staining, Western Blot, Strep-tag, Purification