Journal: PLoS ONE

Article Title: Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys

doi: 10.1371/journal.pone.0253197

Figure Lengend Snippet: A . Immunofluorescent staining for APOL1, nephrin (to identify podocytes), and CD31 (to identify endothelia cells) in the BAC-APOL1 transgenic mouse kidney (G1 mouse is shown). B. Comparison of APOL1 expression patterns in all three APOL1 transgenic lines. Proximal tubules were identified by labeling with fluorescent Lotus tetragonolobus (LT) lectin. APOL1 was present in vascular endothelia (arrow heads), in podocytes (arrows), and trapped in vascular spaces (*), but not tubular epithelia. Scale bar = 40μm.

Article Snippet: FITC-labeled Lotus tetragonolobus lectin (Vector labs) was used to label proximal tubule cells as previously described [ ].

Techniques: Staining, Transgenic Assay, Expressing, Labeling

Journal: PLoS ONE

Article Title: Lack of APOL1 in proximal tubules of normal human kidneys and proteinuric APOL1 transgenic mouse kidneys

doi: 10.1371/journal.pone.0253197

Figure Lengend Snippet: A . Control immunostaining in non-transgenic (wildtype) and proteinuric HIVAN4 mice for APOL1 along with fluorescent Lotus tetragonolobus (“LT”) lectin binding to demarcate the proximal tubule brush border. Since wildtype and HIVAN4 mice do not have APOL1, the staining observed in parietal cells (arrows) is artifact. B . Immunostaining for APOL1 in proteinuric BAC transgenic mice of each APOL1 genotype (representative images are shown, number of animal examined in each genotype group were the same as for ). Images show proximal tubules at the transition with Bowman capsule. The boxed region is magnified below each panel along with the isolated fluorescent channels shown in black and white. C . Positive control immunostaining for a filtered lipoprotein, APOA1, and fluorescently-labelled Lotus tetragonolobus (“LT”) lectin. An APOL1-G0 mouse and an APOL1-G0 x HIVAN4 dual transgenic mouse with proteinuria is shown; below each respective color panel is the individual fluorescent channels (in black and white) of the boxed region for either LT lectin or APOA1. White arrows mark glomerular capillaries containing circulating APOA1 protein within capillary lumens, red arrows denote APOA1 in protein reabsorption droplets at the brush border of proximal tubules. Scale bar = 40μm.

Article Snippet: FITC-labeled Lotus tetragonolobus lectin (Vector labs) was used to label proximal tubule cells as previously described [ ].

Techniques: Immunostaining, Transgenic Assay, Binding Assay, Staining, Isolation, Positive Control

Journal: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

Figure Lengend Snippet:

Article Snippet: L14 , Biotinylated Datura Stramonium Lectin , DSL , Vector Laboratories , BK-3000.

Techniques: Plasmid Preparation, Positive Control

Journal: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

Figure Lengend Snippet:

Article Snippet: L17 , Biotinylated Solanum Tuberosum (Potato) Lectin , STL , Vector Laboratories , BK-3000.

Techniques: Plasmid Preparation, Positive Control

Journal: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

Figure Lengend Snippet:

Article Snippet: L14 , Biotinylated Datura Stramonium Lectin , DSL , Vector Laboratories , BK-3000.

Techniques: Plasmid Preparation, Positive Control

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: Evaluation of the sialylation status of flagellin isolated from PA +Sia (A) The sialic acid specific – Sambucus nigra (SNA) lectin conjugated to agarose beads was used to isolate sialoglycoproteins from PA +Sia lysate (100 µg) by a lectin-based pulldown assay. Highly sialylated bovine salivary mucin (BSM) was used as a positive control. In the case of PA +Sia lysate, the lectin bound sialylated proteins were resolved in 10% SDS-PAGE and visualized by Coomassie Blue staining. Staining revealed several fine bands along with a prominent band near 47 kDa and two bands near 34, 37 kDa. The presence of similar bands in the BSM control indicated that these 34, 37 kDa bands come from the SNA-agarose beads. The band near 47 kDa was excised and processed for in gel trypsin digestion. The protein band was reduced, alkylated then digested by trypsin and subjected to MALDI TOF MS/MS. The generated spectra were searched against a eubacterial protein database by the MASCOT search engine utilizing the Basic Local Alignment Tool (BLAST) of ABI GPS Explorer software, version 3.6. Through peptide mass fingerprinting, the protein band was identified as flagellin. The MS/MS spectra with each tryptic fragments denoted by respective m/z values and the BLAST search results are shown. The peptide fragments within the flagellin sequence which were detected via MALDI TOF MS/MS have been marked with red colour. (B) The isolated, purified Fla +Sia protein (2 μg) resolved to a single band in a 10% SDS-PAGE gel. This single band was carefully excised and subjected to in gel trypsin digestion as before. The peptide digest was analyzed via ESI MS/MS mode by the LTQ Orbitrap mass spectrometer. The generated spectra was searched against the eubacterial protein database compiled from Uniprot using the Sequest HT Proteome Discoverer 1.4 software (Thermo Fisher Scientific) utilizing the MASCOT search engine. All peptide fragments among the flagellin sequence which have been identified in the tryptic digest have been denoted with red colour. (C) Purified Fla +Sia (25 μg) was passively rehydrated into pH 4–7 IPG strips and resolved via isoelectric focussing. The resolved strips were placed over the resolving gel in a10% SDS-PAGE gel and fixed using melted agarose. The protein sample was resolved in the second dimension through electrophoresis. The 2D gels were stained using Coomassie Blue to reveal that the single band protein had split into seven isoforms . (D) The sialic acid content of Fla +Sia /Fla -Sia (500 µg) were estimated and compared using the fluorimetric acetyl acetone method following Shukla et al (1982). Following the acetyl-acetone reaction, sialic acids released from flagellin proteins formed a fluorigenic product which were quantified via a fluorimeter (Ex-410nm, Em-510nm). The amount of sialic acids present in each sample was calculated by comparing with a prepared standard dilution of synthetic N-acetyl Neuraminic acid. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (E) Fla +Sia /Fla -Sia (5μg) resolved via SDS-PAGE was transferred onto PVDF blots via wet transfer method. These blots were probed with biotinylated sialic acid binding lectins – Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA II). The blots were washed and incubated with secondary avidin-HRP and the extent of lectin binding was determined by developing the blot using chemiluminescent substrates. The Ponceau S stained blots are provided to account for equal protein loading. Densitometric data from three independent experiments were used to prepare the bar diagram shown here. Representative blots from one experiment are shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (F) Fla +Sia was used to coat a 96 well ELISA plate. A panel of Siglec-Fc chimera recombinant proteins (Siglec-3,4,5,6,7,9 and 10-Fc) was diluted and incubated in these Fla +Sia coated wells. The wells were then washed and incubated with anti-human IgG Fc-specific peroxidase secondary antibodies. The extent of Siglec-Fc remaining bound to the Fla +Sia coated wells were detected using TMB substrate. The colour which was generated from the bound secondary antibodies was measured as absorbance at 450nm in a plate reader. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (G) As before, Fla +Sia /Fla -Sia (5μg) were resolved in SDS-PAGE, transferred to blots and probed using the same Siglec-Fc panel. The blots were washed and then incubated with secondary anti-human IgG Fc region specific peroxidase antibodies. The extent of Siglec binding was directly visualized by developing the blots in a chemidoc using a chemiluminescent substrate. As before, Ponceau S stained blots are supplied to account for equal loading. One representative blot from three independent experiments performed is shown here.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Isolation, Positive Control, SDS Page, Staining, Control, Tandem Mass Spectroscopy, Generated, Software, Peptide Mass Fingerprinting, Sequencing, Purification, Mass Spectrometry, Electrophoresis, Binding Assay, Incubation, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: (A) Purified Fla +Sia or desialylated Fla +Sia (ds-Fla +Sia ) (20 μg) were passively rehydrated into pH 3-10 IPG strips. The proteins were resolved by isoelectric focusing following the manufacturer’s instructions. These strips were stained by Coomassie blue and then destained before being dried. IPG strips were observed to detect any changes in protein resolution upon sialidase treatment. Representative strips selected from the two independent experiments performed are shown here. (B) As before, Fla +Sia /ds-Fla +Sia (5μg) were resolved in SDS-PAGE, transferred on to PVDF blots and probed with biotinylated sialic acid binding lectins – SNA, MAA II ((5 μg/mL). The blots were washed and then incubated with avidin-HRP. The extent of lectin binding was determined by developing the blots in a chemidoc using a chemiluminescent substrate. Additionally, the galactose specific lectin – peanut agglutinin (PNA) was also used (10 μg/mL). Upon sialidase treatment, the removal of sialic acid residues at the glycan termini exposes the underlying galactose residues. PNA recognizes and binds these exposed galactose residues indicating the successful removal of sialic acids. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (C) In a same set up, Fla +Sia /ds-Fla +Sia blots were probed with Siglec-9-Fc chimera proteins. The blots were washed and then incubated with anti-human IgG Fc specific antibodies. Any change in Siglec-9 interaction with flagellin was detected by developing the blot in a chemidoc. As previously described, Ponceau S stained blots are shown to account for equal protein loading across lanes. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Purification, Staining, SDS Page, Binding Assay, Incubation, Avidin-Biotin Assay

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: Journal of Visualized Experiments : JoVE

Article Title: Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

doi: 10.3791/3791

Figure Lengend Snippet:

Article Snippet: L22 , Biotinylated Jacalin , JACALIN , Vector Laboratories , BK-3000.

Techniques: Plasmid Preparation, Positive Control

Journal: BioTechniques

Article Title: Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay.

doi: 10.2144/000114208

Figure Lengend Snippet: Figure 4. Lectin binding pattern of urinary EVs from healthy subjects. Immobilized uEVs were allowed to react with biotinylated plant lectins for 30 min at RT. The unbound material was washed out, followed by addition of avidin/biotinylated HRPO and TMB solution. Optical density was monitored at 450 nm. Binding of each lectin was normalized to binding of anti-CD63 antibody, which was run as the control for uEV immobilization. The results are mean values of three experiments. uEVs from both male and female donors were analyzed. Error bars represent the standard deviation. Lectins: Con A (specific for high mannose-type, hybrid-type, and biantennary complex type N-glycans), WGA (specific for GlcNAcb1.4 oligomers/Galb1,4GlcNAc), RCA (specific for Galb1,4GlcNAc), SNA (specific for sialic acid a2,6-Gal), MAA (specific for sialic acid a2,3-Gal), PHA-E (specific for bisected biantennary chains), PHA-L (specific for tri/tetraantennary complex-type N-glycans), LCA (specific for bi/triantennary complex-type with core fucose), PNA (specific for Galb1,3GalNAca-Ser/Thr, T antigen), VVL (specific for terminal GalNAca-Ser/Thr, Tn antigen/ GalNAc1,3Gal), DBL (specific for a1,3GalNAc and blood group A antigen).

Article Snippet: Biotinylated plant lectins [concanavalin A (Con A), Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), Maackia amurensis lectin II (MAL II), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA I), Vicia villosa lectin (VVL), and Dolichos biflorus agglutinin (DBA)], VECTASTAIN ABC Kit, biotinylated goat anti-mouse IgG, and biotinylated rabbit anti-goat IgG were purchased from Vector Laboratories (Burlingame, CA).

Techniques: Binding Assay, Avidin-Biotin Assay, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria

doi: 10.3389/fimmu.2022.958952

Figure Lengend Snippet: No binding of 27H8 and M86 to intestinal bacteria in contrast to BSI-B 4. (A) Histograms of flow cytometric analysis of cultured bacterial strains stained with 27H8, M86 and BSI-B 4 and respective isotype controls (IgG1 and IgM). Strains: E coli O86:B7, E.coli BL21, E coli Human Species (HS) and Lactobacillus rhamnosus ( L. rhamnosus ). (B) Dot blot stain of lysed E coli O86:B7 and α-Gal-MSA as positive control. Uncropped blots depicted in Supplementary Figure 1E . (C) Representative histogram blots of flow cytometric analysis of intestinal content (derived from small intestine, cecum and colon) from Ggta1 KO mice (n=3) stained with biotinylated 27H8 and BSI-B 4 , pre-gated for SYBR green positive bacteria ( Supplementary Figure 2A ). (D) Live splenocytes of Ggta1 KO and WT mouse ( Supplementary Figure 2B ) stained with biotinylated 27H8 and BSI-B 4 . (C, D) Control staining with IgG1-biotin isotype and streptavidin-PE (SAv-PE) only.

Article Snippet: 5x10 5 cells were seeded and stained with either 27H8 purified antibody or IgG1κ isotype control (clone: B3102E8, Southern Biotech) at 1 µg/ml, a 1:10 dilution of M86 supernatant, a 1:10 dilution (40 µg/ml) of mouse IgM isotype control (clone: MOPC 104E, Sigma) or a 1:100 dilution of BSI-B 4 conjugated to DyLight ® 594 ( Griffonia simplicifolia isolectin B4, Vector Laboratories, Burlingame, CA, USA) in BSA-buffer.

Techniques: Binding Assay, Cell Culture, Staining, Dot Blot, Positive Control, Derivative Assay, SYBR Green Assay

Journal: Frontiers in Immunology

Article Title: A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria

doi: 10.3389/fimmu.2022.958952

Figure Lengend Snippet: Specificity of 27H8 monoclonal antibody. (A) Dot blot stain of lysed whole blood from a type B blood donor and α-Gal-MSA (positive control) by 27H8, M86 or Lectin (BSI-B 4 ). (B) Screening of 27H8 subcloned hybridoma SN (upper row) and purified 27H8 antibody (middle row) on lysed kidney tissue or cultured kidney cells of wildtype (WT) and GGTA1 knockout (KO) pigs and on WT kidney tissue samples digested with α-Galactosidase (Dig. WT). Further screening molecules: aminopeptidase N (APN) glycosylated with α-Gal, APN only and (α-Gal-containing) bovine thyroglobulin. (A, B) Samples in a row were blotted on one membrane. See Supplementary Figure 1 for uncropped blots. (C) Immunofluorescence microscopy of pig kidney tissue specimens (WT and GGTA1 KO) stained with 27H8 (red) and M86 (green) in the glomerulus region. IgG1 isotype ctrl and secondary antibody only stains (anti-IgG1/anti-IgM) served as controls for fluorescence signal. DNA stained with DAPI (blue). Scale bar (white, left corner): 124.4 µm. (D) Flow cytometry analysis of human embryonic kidney (HEK) cells expressing α-Gal glycosylated APN (upper panel) and APN only (lower panel) stained with 27H8 (red), M86 (green) and BSI-B 4 (blue). Controls: unstained (grey) and Isotype controls (IgG1, IgM, both in black). (A–D) If not otherwise indicated, 27H8 was applied in the purified version.

Article Snippet: 5x10 5 cells were seeded and stained with either 27H8 purified antibody or IgG1κ isotype control (clone: B3102E8, Southern Biotech) at 1 µg/ml, a 1:10 dilution of M86 supernatant, a 1:10 dilution (40 µg/ml) of mouse IgM isotype control (clone: MOPC 104E, Sigma) or a 1:100 dilution of BSI-B 4 conjugated to DyLight ® 594 ( Griffonia simplicifolia isolectin B4, Vector Laboratories, Burlingame, CA, USA) in BSA-buffer.

Techniques: Dot Blot, Staining, Positive Control, Purification, Cell Culture, Knock-Out, Immunofluorescence, Microscopy, Fluorescence, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: Evaluation of the sialylation status of flagellin isolated from PA +Sia (A) The sialic acid specific – Sambucus nigra (SNA) lectin conjugated to agarose beads was used to isolate sialoglycoproteins from PA +Sia lysate (100 µg) by a lectin-based pulldown assay. Highly sialylated bovine salivary mucin (BSM) was used as a positive control. In the case of PA +Sia lysate, the lectin bound sialylated proteins were resolved in 10% SDS-PAGE and visualized by Coomassie Blue staining. Staining revealed several fine bands along with a prominent band near 47 kDa and two bands near 34, 37 kDa. The presence of similar bands in the BSM control indicated that these 34, 37 kDa bands come from the SNA-agarose beads. The band near 47 kDa was excised and processed for in gel trypsin digestion. The protein band was reduced, alkylated then digested by trypsin and subjected to MALDI TOF MS/MS. The generated spectra were searched against a eubacterial protein database by the MASCOT search engine utilizing the Basic Local Alignment Tool (BLAST) of ABI GPS Explorer software, version 3.6. Through peptide mass fingerprinting, the protein band was identified as flagellin. The MS/MS spectra with each tryptic fragments denoted by respective m/z values and the BLAST search results are shown. The peptide fragments within the flagellin sequence which were detected via MALDI TOF MS/MS have been marked with red colour. (B) The isolated, purified Fla +Sia protein (2 μg) resolved to a single band in a 10% SDS-PAGE gel. This single band was carefully excised and subjected to in gel trypsin digestion as before. The peptide digest was analyzed via ESI MS/MS mode by the LTQ Orbitrap mass spectrometer. The generated spectra was searched against the eubacterial protein database compiled from Uniprot using the Sequest HT Proteome Discoverer 1.4 software (Thermo Fisher Scientific) utilizing the MASCOT search engine. All peptide fragments among the flagellin sequence which have been identified in the tryptic digest have been denoted with red colour. (C) Purified Fla +Sia (25 μg) was passively rehydrated into pH 4–7 IPG strips and resolved via isoelectric focussing. The resolved strips were placed over the resolving gel in a10% SDS-PAGE gel and fixed using melted agarose. The protein sample was resolved in the second dimension through electrophoresis. The 2D gels were stained using Coomassie Blue to reveal that the single band protein had split into seven isoforms . (D) The sialic acid content of Fla +Sia /Fla -Sia (500 µg) were estimated and compared using the fluorimetric acetyl acetone method following Shukla et al (1982). Following the acetyl-acetone reaction, sialic acids released from flagellin proteins formed a fluorigenic product which were quantified via a fluorimeter (Ex-410nm, Em-510nm). The amount of sialic acids present in each sample was calculated by comparing with a prepared standard dilution of synthetic N-acetyl Neuraminic acid. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (E) Fla +Sia /Fla -Sia (5μg) resolved via SDS-PAGE was transferred onto PVDF blots via wet transfer method. These blots were probed with biotinylated sialic acid binding lectins – Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA II). The blots were washed and incubated with secondary avidin-HRP and the extent of lectin binding was determined by developing the blot using chemiluminescent substrates. The Ponceau S stained blots are provided to account for equal protein loading. Densitometric data from three independent experiments were used to prepare the bar diagram shown here. Representative blots from one experiment are shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (F) Fla +Sia was used to coat a 96 well ELISA plate. A panel of Siglec-Fc chimera recombinant proteins (Siglec-3,4,5,6,7,9 and 10-Fc) was diluted and incubated in these Fla +Sia coated wells. The wells were then washed and incubated with anti-human IgG Fc-specific peroxidase secondary antibodies. The extent of Siglec-Fc remaining bound to the Fla +Sia coated wells were detected using TMB substrate. The colour which was generated from the bound secondary antibodies was measured as absorbance at 450nm in a plate reader. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (G) As before, Fla +Sia /Fla -Sia (5μg) were resolved in SDS-PAGE, transferred to blots and probed using the same Siglec-Fc panel. The blots were washed and then incubated with secondary anti-human IgG Fc region specific peroxidase antibodies. The extent of Siglec binding was directly visualized by developing the blots in a chemidoc using a chemiluminescent substrate. As before, Ponceau S stained blots are supplied to account for equal loading. One representative blot from three independent experiments performed is shown here.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Isolation, Positive Control, SDS Page, Staining, Control, Tandem Mass Spectroscopy, Generated, Software, Peptide Mass Fingerprinting, Sequencing, Purification, Mass Spectrometry, Electrophoresis, Binding Assay, Incubation, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: (A) Purified Fla +Sia or desialylated Fla +Sia (ds-Fla +Sia ) (20 μg) were passively rehydrated into pH 3-10 IPG strips. The proteins were resolved by isoelectric focusing following the manufacturer’s instructions. These strips were stained by Coomassie blue and then destained before being dried. IPG strips were observed to detect any changes in protein resolution upon sialidase treatment. Representative strips selected from the two independent experiments performed are shown here. (B) As before, Fla +Sia /ds-Fla +Sia (5μg) were resolved in SDS-PAGE, transferred on to PVDF blots and probed with biotinylated sialic acid binding lectins – SNA, MAA II ((5 μg/mL). The blots were washed and then incubated with avidin-HRP. The extent of lectin binding was determined by developing the blots in a chemidoc using a chemiluminescent substrate. Additionally, the galactose specific lectin – peanut agglutinin (PNA) was also used (10 μg/mL). Upon sialidase treatment, the removal of sialic acid residues at the glycan termini exposes the underlying galactose residues. PNA recognizes and binds these exposed galactose residues indicating the successful removal of sialic acids. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (C) In a same set up, Fla +Sia /ds-Fla +Sia blots were probed with Siglec-9-Fc chimera proteins. The blots were washed and then incubated with anti-human IgG Fc specific antibodies. Any change in Siglec-9 interaction with flagellin was detected by developing the blot in a chemidoc. As previously described, Ponceau S stained blots are shown to account for equal protein loading across lanes. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Purification, Staining, SDS Page, Binding Assay, Incubation, Avidin-Biotin Assay