Journal: Journal of the American Chemical Society

Article Title: The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O ‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O ‑Glucose

doi: 10.1021/jacs.7b13488

Figure Lengend Snippet: A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O-GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac46AzGlc (200 μM) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β-actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β-elimination. NIH3T3 cells were treated with either Ac46AzGlc (200 μM) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H2O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac46AzGlc (200 μM) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H2O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

Article Snippet: The blots washed three times in TBST for 5 min and incubated with biotin-conjugated Concanavalin A (Vector Lab), diluted 1:1000 in TBST, for 1 h. The blot was then washed 3× with TBST for 10 min. Then the blot was then incubated with Strep-HRP at 1:1000 in blocking buffer for 1 h. After being washed 3× with TBST for 10 min, the blot was developed using ECL reagents.

Techniques: Labeling, Western Blot, Negative Control, SDS Page, Incubation, Fluorescence

Journal: Scientific Reports

Article Title: Targeting cell-derived markers to improve the detection of invisible biological traces for the purpose of genetic-based criminal identification

doi: 10.1038/s41598-023-45366-y

Figure Lengend Snippet: The principal carbohydrate patterns recognized by the 4 lectins tested.

Article Snippet: Samples were incubated for 20 min in a humidity chamber using a biotinylated lectin solution (at final concentration 10 µg/ml): Concanavalin A (ConA), Peanut agglutinin (PNA), Elderberry lectin (SNA), and Ulex europaeus agglutinin (UEA) (B-1005, B-1075, B-1305, and B-1065 respectively, Vector Laboratories, USA).

Techniques:

Journal: bioRxiv

Article Title: The restricted nature of protein glycosylation in the mammalian brain

doi: 10.1101/2020.10.01.322537

Figure Lengend Snippet: Protein lysate from mouse cortex and cerebellum with human plasma as a positive control was treated with or without PNGase F and visualized using biotinylated lectins (Con A, GNL, PHA-E, AAL, RCA, and SNA) in addition to immunoblotting for actin and staining for total protein. Non-specific binding of lectins to PNGase F is noted by an asterisk (*) near 35 kDa. A schematic with common lectin-binding sites is shown for reference.

Article Snippet: Membranes were then incubated in 5% BSA in TBS-Tween 0.1% for 1 hour, followed by incubation with biotinylated lectins (Vector Labs: AAL B-1395, SNA B-1305, GNL B-1245, PHA-E B-1125, RCA B-1085, Con A B-1105) at a 1:1,000 dilution (1:20,000 for Con A) and 1:2,000 dilution of mouse anti-actin antibody (Abcam, ab8226) in 5% BSA in TBS-Tween 0.1%, overnight at 4°C on a rocking platform shaker.

Techniques: Positive Control, Western Blot, Staining, Binding Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Knockdown of INPP5K compromises the differentiation of N2A cells

doi: 10.3389/fnmol.2024.1356343

Figure Lengend Snippet: Knockdown of INPP5K in N2A cells interferes with protein glycosylation. (A) Representative Coomassie stained membranes and membranes probed with different biotinylated lectins, i.e., WGA, RCAI, SNL, MAL, PNA, LCH, and Con A, and the respective quantifications. GAPDH and Coomassie staining served as a loading control. Green circle = mannose, yellow circle = galactose, blue square = N -acetylglucosamine, yellow square = N -acetylgalactosamine, violet diamond = N-glycolylneuraminic acid, and red triangle = fucose. Lectin signals were normalized to GAPDH. N2A cells were transfected with either scRNA or siRNA to knock down INPP5K, harvested, and the respective protein lysates separated by SDS-PAGE and transferred to membranes ( n = 3 experiments, Student's t -test). (B) Staining of N2A cells transfected with either scRNA or siRNA to knock down INPP5K with WGA, anti-PDI, anti-INPP5K, and DAPI to label nuclei (scale bar 10 μm, n = 4 experiments with 200–5,000 cells per condition and experiment, Student's t -test). White arrowheads indicate examples of cells with WGA-positive deposits co-labeling with the ER marker PDI. Cells with intracellular deposits labeled by WGA were quantified. (C) As a control, we also incubated N2A cells with either secondary antibodies alone or fluorophore-coupled streptavidin, which was used to detect biotinylated WGA (scale bar 10 μm). Quantitative data are presented as mean ± SEM with individual data points. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: The following primary antibodies or lectins were used: rabbit anti-INPPK (Proteintech) 1:500, rabbit anti-GAPDH (Proteintech) 1:1,000, biotin WGA (Biozol) 1:300, biotin PNA (Bioworld) 1:300, biotin SNL (Biozol) 1:300, biotin RCAI (Biozol) 1:300, biotin Con A (Biozol) 1:300, biotin LCH (Ey Laboratories) 1:300, and biotin MAL (Biomol) 1:300.

Techniques: Staining, Transfection, SDS Page, Labeling, Marker, Incubation