fluorescein conjugated lectins  (Vector Laboratories)

Journal: The Journal of Biological Chemistry

Article Title: Antibodies That Detect O-Linked β-d-N-Acetylglucosamine on the Extracellular Domain of Cell Surface Glycoproteins *

doi: 10.1074/jbc.M113.492512

Figure Lengend Snippet: CTD110.6 recognizes both O- GlcNAc and terminal β-GlcNAc on cell surface glycoproteins. A , N- ), of swainsonine. B , binding of L-PHA ( upper ) and mAb CTD110.6 ( lower ) to fixed cells. Lec2 and Lec8 cells were cultured with or without 5 μg/ml swainsonine ( SW ) for 4 days. Cells (4 × 10 5 ) were incubated with 20 μg/ml fluorescein-labeled L-PHA or 40 μg/ml CTD110.6 on ice for 20 min followed by 10 μg/ml Cy5-conjugated anti-mouse IgM antibody. Gray profiles , secondary Abs; thin line , control cells; bold line , swainsonine-treated cells. Representative results are from two independent experiments.

Article Snippet: Washed cells were incubated with 50 μl of binding buffer containing 1 μg of fluorescein-labeled L-PHA ( Phaseolus vulgaris leukoagglutinin; Vector, Burlingame, CA) or 2 μg of CTD110.6 mAb, followed by incubation with 50 μl of binding buffer containing 0.5 μg of Cy5-conjugated anti-mouse IgM.

Techniques: Binding Assay, Cell Culture, Incubation, Labeling

Journal: The Journal of Cell Biology

Article Title: A testis-specific regulator of complex and hybrid N-glycan synthesis

doi: 10.1083/jcb.201004102

Figure Lengend Snippet: GlcNAcT-I-KDEL localized mainly in the ER is active. Lec1 cells stably expressing empty vector, GlcNAcT-I-HA-KDEL, or GlcNAcT-I-HA were analyzed for (A) Con A resistance and (B) L-PHA-FITC binding by flow cytometry. (C) GlcNAcT-I and β4GalT activities in transfectant lysates were determined. Bars represent range ( n = 2). (D) HeLa cells transiently expressing GlcNAcT-I-HA-KDEL were fixed and immunofluorescently labeled for GlcNAcT-I-HA-KDEL (red) and endogenous ManII (green). Bars, 10 µm.

Article Snippet: For cell sorting, cells were washed with FACS binding buffer without sodium azide and incubated in the same buffer containing 12 µg/ml GNA-FITC or L-PHA-FITC.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Binding Assay, Flow Cytometry, Cytometry, Transfection, Labeling

Journal: bioRxiv

Article Title: Overexpression of CD47 is associated with brain overgrowth in 16p11.2 deletion syndrome

doi: 10.1101/808022

Figure Lengend Snippet: Calreticulin, a pro-phagocytic signal, is upregulated in 16p11.2 deletion NPCs and OPCs. A. Representative histograms show the mean fluorescence intensities (MFI) of cell-surface expression of Calreticulin (CRT) in NPCs derived from control, 16p_del, and 16p_dup lines. B. Quantification of CRT MFI of cell-surface expression of CRT in the 16p_del NPCs relative to 16p_dup and control NPCs. C. Quantification of Phaseolus Vulgaris Leucoagglutinin (PHA-L), indicative of asialoglycan binding sites for CRT, in NPCs in 16p_deletion lines relative to control and 16p_duplication lines. D. Representative histograms show the MFI of cell-surface expression of CRT in OPCs derived from control, 16p_del, and 16p_dup lines. E. Quantification of CRT MFI of cell-surface expression of CRT in the 16p_del OPCs relative to 16p_dup and control OPCs. F. Quantification of PHA-L in OPCs in 16p_deletion lines relative to control and 16p_duplication lines. n=2 biological replicates per cell line (16p_dup, n=2; control, n=3; 16p_del, n=4 cell lines). All data are mean ± SE; P values were determined by one-way ANOVA followed by post-hoc Tukey HSD Test.

Article Snippet: The lectin fluorescein labeled Phaseolus Vulgaris Leucoagglutinin (PHA-L) (1:200; Vector Laboratories) was used to stain for PHA-L. For O4 staining, the cells were first incubated in primary antibody mouse-anti-O4 antibody (Clone 81; 1:100; EMD millipore), followed by secondary antibody incubation, anti-Mouse IgM Alexa Fluor 488 (1:1000; Life Technologies).

Techniques: Fluorescence, Expressing, Derivative Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The Role of cis Dimerization of Signal Regulatory Protein ? (SIRP?) in Binding to CD47 *

doi: 10.1074/jbc.M110.180018

Figure Lengend Snippet: SIRPα dimerization is disrupted after treatment with tunicamycin. A , flow cytometric analysis of tunicamycin-treated ( solid line ) and untreated ( dashed line ) CHO-SIRPα transfectants labeled with PHA-L-FITC ( top left panel ) to measure the

Article Snippet: Cells were grown to confluence and subjected to cross-linking analyses and flow cytometric analyses using phytohemagglutinin type L conjugated with FITC (PHA-L-FITC; Vector Laboratories) to assess the reduction in N -linked glycosylation.

Techniques: Flow Cytometry, Labeling