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: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) Schematic diagram of the potential N -glycosylation sites (N X (S/T)) on the ß4 integrin subunit. The sites corresponding to the putative N -glycosylation sites on the ß4 integrin subunit (Asn 327 , Asn 491 , Asn 579 , Asn 617 , and Asn 695 ) are shown by flags. Numbers and boxes indicate the number of amino acid residue and the four intracellular fibronectin type III repeats, respectively. TM, transmembrane region. (B) Cell lysates from normal keratinocytes were immunoprecipitated using a control IgG or an anti-ß4 integrin Ab. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the biotinylated L 4 -PHA lectin (upper left panel) and E 4 -PHA lectin (upper right panel) or anti-ß4 and anti-α6 integrin Abs (lower panels). IB, immunoblot. The black and white arrowheads indicate the ß4 integrin and α6 integrin subunits, respectively. Ordinates indicate molecular sizes in kDa of marker proteins. (C) Cell morphology of the WT and ΔNß4 integrin-expressing keratinocytes during cell culture. (D) The cell-spreading area in A was calculated using computer software (Image J). Each bar represents the mean ± S.D. of triplicate assays. *p<0.001 (unpaired t-test vs.WT). (E) 20 µg of cell lysates from control lacZ- (lac), WTß4, ΔNß4 integrin-expressing keratinocytes were run on a 6% gel under reducing conditions, blotted onto a nitrocellulose membrane, and then probed with an anti-ß4 integrin Ab. (F) Cell lysates were immunoprecipitated using a polyclonal Ab against ß4 integrin. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the indicated biotinylated lectins or an anti-ß4 integrin Ab. (G) Cell surface expression levels of α3, α6, ß1 and ß4 integrin subunits of WT or ΔNß4 integrin-expressing keratinocytes were examined using FACS analysis. Prior to analysis, cells were incubated with either the indicated integrin Abs or control IgG, followed by incubation with Alexa Fluor conjugated secondary Abs, as described under “ .” (H) Immunoprecipitates from WT or ΔN keratinocytes with an anti-α6 integrin were probed with an anti-ß4 (upper panel) or ß1 integrin (lower panel) Ab.

Article Snippet: Biotinylated DSA, L 4 -PHA, E 4 -PHA, ConA, SSA and MAM lectins were from Seikagaku Biobusiness Corporation (Tokyo, Japan).

Techniques: Glycoproteomics, Residue, Immunoprecipitation, Control, Western Blot, Marker, Expressing, Cell Culture, Software, Membrane, Incubation

Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) Cell surface expression levels of EGFR by FACS analysis against WT and ΔN keratinocytes. (B) Cell lysates from lac, WT and ΔN keratinocytes were immunoprecipitated using an anti-EGFR pAb. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and were probed with either the biotinylated E 4 -PHA lectin, L 4 -PHA lectin, DSA lectin and ConA lectin or an anti-EGFR Ab. (C) Covalent cross-linking was performed on WT and ΔN keratinocytes. After cross-linking, collected cell lysates were immunoprecipitated using an anti-EGFR pAb. Immunoprecipitates were run on a 5–20% SDS-polyacrylamide gel and were probed with the indicated Abs.

Article Snippet: Biotinylated DSA, L 4 -PHA, E 4 -PHA, ConA, SSA and MAM lectins were from Seikagaku Biobusiness Corporation (Tokyo, Japan).

Techniques: Expressing, Immunoprecipitation

Journal: BMC Biotechnology

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology

doi: 10.1186/s12896-014-0101-0

Figure Lengend Snippet: MVN binds high-mannose type N -glycans on Pp -hGGT1. Purified Pp -hGGT1 was incubated in the absence or presence of PNGaseF. Pp -hGGT1 (0.1 μg) from each reaction was resolved by SDS-PAGE, electroblotted onto nitrocellulose, and probed with either an antibody against the large subunit of hGGT1 (left panel) or with biotinylated-MVN (right panel). MW , molecular weight markers.

Article Snippet: Each membrane was incubated in PBST with either 1.0 μg/mL biotinylated MVN (described herein), 1.0 μg/mL biotinylated Datura stramonium lectin (DSL, Vector Labs), or 0.2 μg/mL biotinylated Phaseolus vulgaris -Erythroagglutinin (Pha-E, Vector Labs) for 1 h at room temperature.

Techniques: Purification, Incubation, SDS Page, Molecular Weight

Journal: BMC Biotechnology

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology

doi: 10.1186/s12896-014-0101-0

Figure Lengend Snippet: Antibody lectin sandwich assay (ALSA). (A) Graphical depiction of ALSA strategy. The hGGT1 antibodies are printed on a nitrocellulose slide and chemically derivatized to inactivate their glycans. hGGT1 is captured by the immobilized antibody and N -glycan features are probed using biotinylated lectins, which are subsequently detected using streptavidin-phycoerythrin and fluorescence scanning. (B) One printed antibody array well is shown with the magnified capture antibodies. Individual hGGT1 capture spots have been cut out to show triplicate detection of Datura stramonium lectin (DSL) among 64-fold kidney sample dilutions (equivalent to ~75 ng of total extracted kidney protein). (C) Representative results for hGGT1 capture antibody and detection reagents (indicated in the column labels).

Article Snippet: Each membrane was incubated in PBST with either 1.0 μg/mL biotinylated MVN (described herein), 1.0 μg/mL biotinylated Datura stramonium lectin (DSL, Vector Labs), or 0.2 μg/mL biotinylated Phaseolus vulgaris -Erythroagglutinin (Pha-E, Vector Labs) for 1 h at room temperature.

Techniques: Fluorescence, Ab Array

Journal: BMC Biotechnology

Article Title: Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology

doi: 10.1186/s12896-014-0101-0

Figure Lengend Snippet: hGGT1 lectin blotting confirms differential ALSA binding affinities. Membrane extracts from normal human kidney and liver tissue or Pichia pastoris -expressed hGGT1 were activity-normalized and subjected to immunoprecipitation with a polyclonal anti-hGGT1 large subunit antibody. Equal volumes from each immunoprecipitation eluate were resolved by SDS-PAGE and affinity blotted with anti-hGGT1 ( hGGT1 , top panel ) or the biotinylated lectins, microvirin ( MVN , second panel ), Phaseolus vulgaris Erythroagglutinin ( Pha - E , third panel ), and Datura stramonium lectin ( DSL , bottom panel ). Expanded views of the immunoblots shown here along with the relevant molecular weight markers are included in Additional file .

Article Snippet: Each membrane was incubated in PBST with either 1.0 μg/mL biotinylated MVN (described herein), 1.0 μg/mL biotinylated Datura stramonium lectin (DSL, Vector Labs), or 0.2 μg/mL biotinylated Phaseolus vulgaris -Erythroagglutinin (Pha-E, Vector Labs) for 1 h at room temperature.

Techniques: Binding Assay, Activity Assay, Immunoprecipitation, SDS Page, Western Blot, Molecular Weight