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: Brain Pathology

Article Title: Microvessel stenosis, enlarged perivascular spaces, and fibrinogen deposition are associated with ischemic periventricular white matter hyperintensities

doi: 10.1111/bpa.13017

Figure Lengend Snippet: Increases in microglia are seen in the periventricular and subcortical white matter and are associated with fibrinogen uptake. (A) Example images of specimens with high and low levels of microglia, stained with RCA Lectin. (B) Microglia were counted within the periventricular and subcortical white matter (data points represent the average count for 3 images per region) in specimens with none, mild and moderate pvWMH or evidence of PVI. (C) Microglia count within the periventricular and subcortical white matter subjects with fibrinogen ratings of 1–4 in either the periventricular or subcortical white matter. Microglia count is associated with both periventricular and subcortical fibrinogen ratings. Comparison performed using one‐way ANOVA with Tukey's post‐hoc analysis, significance value of p = 0.05. (D) Example images taken from the subcortical white matter of a specimen with high fibrinogen rating and high microglial count. All images captured at 20x magnification; scale bar indicates 100 μm

Article Snippet: Tissue was stained with Ricinus communis ‐I (RCA) Lectin (Vector Labs, Brockville, Canada, 1:10,000) prior to Movat's pentachrome staining, the slides were placed in 0.5% periodic acid for 15′ caused by over‐fixation.

Techniques: Staining

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