wfa  (Vector Laboratories)


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    Name:
    Unconjugated Wisteria Floribunda Lectin WFA WFL
    Description:
    Wisteria floribunda lectin WFA WFL The binding specificity of Wisteria floribunda lectin WFL is not completely clear but this lectin appears to preferentially bind carbohydrate structures terminating in N acetylgalactosamine linked α or β to the 3 or 6 position of galactose This lectin has been used to fractionate lymphocyte populations and although not mitogenic elicits the production of lymphokines from murine splenocytes
    Catalog Number:
    l-1350
    Price:
    None
    Category:
    Proteins
    Size:
    5 mg
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    Structured Review

    Vector Laboratories wfa
    Unconjugated Wisteria Floribunda Lectin WFA WFL
    Wisteria floribunda lectin WFA WFL The binding specificity of Wisteria floribunda lectin WFL is not completely clear but this lectin appears to preferentially bind carbohydrate structures terminating in N acetylgalactosamine linked α or β to the 3 or 6 position of galactose This lectin has been used to fractionate lymphocyte populations and although not mitogenic elicits the production of lymphokines from murine splenocytes
    https://www.bioz.com/result/wfa/product/Vector Laboratories
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    wfa - by Bioz Stars, 2021-03
    93/100 stars

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    Related Articles

    Staining:

    Article Title: Extracellular Molecular Markers and Soma Size of Inhibitory Neurons: Evidence for Four Subtypes of GABAergic Cells in the Inferior Colliculus
    Article Snippet: One series was stained for brain nitric oxide synthase (bNOS) to identify IC subdivisions, and one or more remaining series were stained with a four-color immunofluorescence procedure to analyze GABAergic and non-GABAergic cells. .. For four-color staining, tissue sections were washed in PBS (0.9% NaCl in 0.01 m phosphate buffer) and then permeablized in 0.2% Triton X-100 in PBS for 30 min. Nonspecific staining was blocked by treating sections with a 10% normal goat serum (NGS) solution containing 0.1% Triton X-100 in PBS for 1 h. After blocking, PNs were stained with fluorescein-labeled Wisteria floribunda agglutinin in PBS for 1 h (WFA; 1:100; Vector Laboratories). .. Sections were rinsed with PBS, and then a mixture of primary antibodies was applied in a solution containing 1% NGS and 0.2% Triton X-100.

    Article Title: Pseudomonas aeruginosa and Candida albicans both accumulate greater biomass in dual species biofilms under flow
    Article Snippet: A 458-nm laser line was used to excite Wisteria floribunda lectin stain in the case of Pel quantification experiments. .. A 458-nm laser line was used to excite Wisteria floribunda lectin stain in the case of Pel quantification experiments. .. All quantitative analysis of microscopy data was performed using BiofilmQ .

    Article Title: Distinct neural circuits for the formation and retrieval of episodic memories
    Article Snippet: After three more wash steps of 10 min each in PBS-T, slices were mounted on microscope slides. .. Antibodies used for staining were as follows: CA1-specific excitatory neurons were stained with rabbit anti-WFS1 (1:400, Proteintech) and anti-rabbit Alexa-555 (1:500), excitatory neurons were stained with mouse anti-CaMKII (1:200, Abcam) and anti-mouse Alexa-555 (1:300), inhibitory neurons were stained with mouse anti-GAD67 (1:500, Millipore) and anti-mouse Alexa-555 (1:300), nuclei were stained with DAPI (1:3000, Sigma), neuronal nuclei were stained with mouse anti-NeuN (1:200, Millipore) and anti-mouse Alexa-555 (1:300), parvalbumin inhibitory neurons were stained with mouse anti-PV (1:500, Swant) and anti-mouse Alexa-555 (1:300), Wisteria floribunda lectin was stained with biotinylated WFA lectin (1:3000, Vector Labs) and streptavidin Alexa-555 (1:200), calbindin was stained with mouse anti-CALB (1:500, Swant) and anti-mouse Alexa-555 (1:300), vesicular glutamate transporter 1 was stained with rabbit anti-VGLUT1 (1:1000, Synaptic Systems) and anti-rabbit Alexa-488 (1:300), vesicular glutamate transporter 2 was stained with rabbit anti-VGLUT2 (1:500, Synaptic Systems) and anti-rabbit Alexa-488 (1:300), vesicular glutamate transporter 3 was stained with guinea pig anti-VGLUT3 (1:1000, Millipore) and anti-guinea pig Alexa-488 (1:500), myelin basic protein (MBP) was stained with rabbit anti-MBP (1:1000, Abcam) and anti-rabbit Alexa-546, cFos was stained with rabbit anti-cFos (1:400, Santa Cruz Biotechnology) and anti-rabbit Alexa-488, and TRE-ChR2-eYFP ( ) was stained with chicken anti-GFP (1:1000, Life Technologies) and anti-chicken Alexa-633. .. Mice were anesthetized with 750–1000 mg kg−1 avertin and transcardially perfused with a hydrogel monomer solution containing 4% acrylamide, 0.05% bis-acrylamide, 0.25% VA-044 initiator, and 4% PFA in PBS.

    Article Title: Sialylation regulates myofibroblast differentiation of human skin fibroblasts
    Article Snippet: We used the following primary antibodies: monoclonal mouse anti-CD44 (dilution 1:100; R & D Systems Inc., Minneapolis, MN, USA), polyclonal rabbit anti-epidermal growth factor receptor (EGFR; dilution 1:100; GeneTex Inc., Irvine, CA, USA), and polyclonal rabbit anti-NEU1 (dilution 1:100; Thermo Fisher Scientific, Waltham, MA, USA). .. For lectin staining, FITC-conjugated Wisteria floribunda agglutinin (WFA; dilution 1:100; Vector Laboratories, Peterborough, UK), FITC-conjugated peanut agglutinin (PNA; dilution 1:100; J-Oil Mills, Tokyo, Japan), biotin-conjugated Maackia amurensis lectin II (MAL-II; dilution 1:100; Vector Laboratories), biotin-conjugated Erythrina cristagalli agglutinin (ECA; dilution 1:100; J-Oil Mills), and FITC-streptavidin (dilution 1:100; BioLegend, San Diego, CA, USA) were used. .. Mean fluorescence intensities (MFIs) were calculated by subtracting the intensities of the controls.

    Next-Generation Sequencing:

    Article Title: Extracellular Molecular Markers and Soma Size of Inhibitory Neurons: Evidence for Four Subtypes of GABAergic Cells in the Inferior Colliculus
    Article Snippet: One series was stained for brain nitric oxide synthase (bNOS) to identify IC subdivisions, and one or more remaining series were stained with a four-color immunofluorescence procedure to analyze GABAergic and non-GABAergic cells. .. For four-color staining, tissue sections were washed in PBS (0.9% NaCl in 0.01 m phosphate buffer) and then permeablized in 0.2% Triton X-100 in PBS for 30 min. Nonspecific staining was blocked by treating sections with a 10% normal goat serum (NGS) solution containing 0.1% Triton X-100 in PBS for 1 h. After blocking, PNs were stained with fluorescein-labeled Wisteria floribunda agglutinin in PBS for 1 h (WFA; 1:100; Vector Laboratories). .. Sections were rinsed with PBS, and then a mixture of primary antibodies was applied in a solution containing 1% NGS and 0.2% Triton X-100.

    Blocking Assay:

    Article Title: Extracellular Molecular Markers and Soma Size of Inhibitory Neurons: Evidence for Four Subtypes of GABAergic Cells in the Inferior Colliculus
    Article Snippet: One series was stained for brain nitric oxide synthase (bNOS) to identify IC subdivisions, and one or more remaining series were stained with a four-color immunofluorescence procedure to analyze GABAergic and non-GABAergic cells. .. For four-color staining, tissue sections were washed in PBS (0.9% NaCl in 0.01 m phosphate buffer) and then permeablized in 0.2% Triton X-100 in PBS for 30 min. Nonspecific staining was blocked by treating sections with a 10% normal goat serum (NGS) solution containing 0.1% Triton X-100 in PBS for 1 h. After blocking, PNs were stained with fluorescein-labeled Wisteria floribunda agglutinin in PBS for 1 h (WFA; 1:100; Vector Laboratories). .. Sections were rinsed with PBS, and then a mixture of primary antibodies was applied in a solution containing 1% NGS and 0.2% Triton X-100.

    Incubation:

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration
    Article Snippet: .. sWGA and WFA enrichment of protein lysates 3.5-mg protein samples were incubated with 1.2 ml sWGA-conjugated agarose slurry (AL-1023S; Vector laboratories) or WFA-conjugated agarose slurry (AL-1353; Vector Laboratories) and gently rotated overnight at 4°C. sWGA and WFA agarose was washed four times in RIPA or standard DGC buffer ( ) containing fresh protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mM benzamidine, 0.2 mM PMSF, 5 µM calpain I, and 5 µM calpeptin) to remove unbound proteins. .. Bound proteins were eluted with 0.3 M GlcNAc (sWGA) or 0.3 M GalNAc (WFA; Sigma-Aldrich) and concentrated using filtration columns (Centricon Ultracel; Millipore) by centrifugation at 4,000 g for 20 min.

    Binding Assay:

    Article Title: Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain
    Article Snippet: For brevican and EAV(M)ESE immunoblotting, the membranes were washed with Buffer B (10 mM phosphate buffered saline, pH 7.4 containing 0.05% Tween 20) for 5 minutes, blocked for 1 h in 5% non-fat dry milk diluted in Buffer B and probed for 2 hours using primary antibodies against mouse anti-brevican (1:1000, BD Transduction Labs, San Jose, CA), rabbit anti-EAMESE (1:1000) [ ] (the neoepitope sequence for mouse brevican fragment), or rabbit anti-EAVESE (1:500) [ , ] (the neoepitope sequence for rat brevican fragment). .. For WFA blotting, the membranes were washed with Buffer B for 5 minutes, blocked in 1% bovine serum albumin diluted in Buffer B for 1 hour and probed for 2 hours using biotinylated Wisteria floribunda lectin (1:10,000 in 1% BSA, Vector Laboratories, Burlingame, CA) as the primary binding reagent. .. Primary antibodies and biotinylated Wisteria floribunda lectin were detected with corresponding secondary antibodies including anti-mouse, anti-rabbit and streptavidin conjugated to horse radish peroxidase (Chemicon, Temecula, CA), respectively.

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    • wfa enrichment  (Vector Laboratories)
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    Vector Laboratories wfa enrichment
    SSPN is required for activation of Akt signaling. (A) Quadriceps protein lysates were prepared in modified RIPA buffer, and 50-µg samples were analyzed by immunoblotting as shown. GAPDH and Coomassie blue (CB) staining are provided as controls for equal loading. (B) Total skeletal muscle from wild-type (WT) and SSPN-null (SSPN −/− ) mice were solubilized in modified RIPA buffer, and 60 µg of each sample was analyzed by immunoblotting with indicated antibodies. GAPDH is provided as a control for equal loading. (C) Skeletal muscle protein lysates from WT, mdx , utrophin-deficient mdx muscle ( mdx :utr −/− ), and α7 integrin–deficient mdx muscle ( mdx : α7 −/− ) were enriched by <t>sWGA</t> lectin affinity chromatography (sWGA eluate). Immunoblots of 10-µg bound proteins eluted with GlcNAc are shown. Void (unbound) fractions are shown in Fig. S5 B . (D) WT quadriceps were injected with CTX to induce skeletal muscle degeneration/regeneration to evaluate the expression of utrophin and <t>WFA</t> binding without the complications of mdx pathology. Injected muscle cryosections were costained with utrophin (green fluorescence) and embryonic myosin heavy chain (eMHC; red fluorescence) as a marker for newly regenerated fibers 4 d after CTX injection. Serial sections were stained with WFA lectin (green fluorescence). Staining was visualized by indirect immunofluorescence. Actively regenerating myofibers displayed robust utrophin expression and WFA binding around the same nonjunctional areas of the sarcolemma. Mice were 6.5 wk of age at the time of analysis. Bar, 50 μm. Utr, utrophin; Dys, dystrophin; P, phospho; Intg, integrin.
    Wfa Enrichment, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wfa enrichment/product/Vector Laboratories
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    SSPN is required for activation of Akt signaling. (A) Quadriceps protein lysates were prepared in modified RIPA buffer, and 50-µg samples were analyzed by immunoblotting as shown. GAPDH and Coomassie blue (CB) staining are provided as controls for equal loading. (B) Total skeletal muscle from wild-type (WT) and SSPN-null (SSPN −/− ) mice were solubilized in modified RIPA buffer, and 60 µg of each sample was analyzed by immunoblotting with indicated antibodies. GAPDH is provided as a control for equal loading. (C) Skeletal muscle protein lysates from WT, mdx , utrophin-deficient mdx muscle ( mdx :utr −/− ), and α7 integrin–deficient mdx muscle ( mdx : α7 −/− ) were enriched by sWGA lectin affinity chromatography (sWGA eluate). Immunoblots of 10-µg bound proteins eluted with GlcNAc are shown. Void (unbound) fractions are shown in Fig. S5 B . (D) WT quadriceps were injected with CTX to induce skeletal muscle degeneration/regeneration to evaluate the expression of utrophin and WFA binding without the complications of mdx pathology. Injected muscle cryosections were costained with utrophin (green fluorescence) and embryonic myosin heavy chain (eMHC; red fluorescence) as a marker for newly regenerated fibers 4 d after CTX injection. Serial sections were stained with WFA lectin (green fluorescence). Staining was visualized by indirect immunofluorescence. Actively regenerating myofibers displayed robust utrophin expression and WFA binding around the same nonjunctional areas of the sarcolemma. Mice were 6.5 wk of age at the time of analysis. Bar, 50 μm. Utr, utrophin; Dys, dystrophin; P, phospho; Intg, integrin.

    Journal: The Journal of Cell Biology

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

    doi: 10.1083/jcb.201110032

    Figure Lengend Snippet: SSPN is required for activation of Akt signaling. (A) Quadriceps protein lysates were prepared in modified RIPA buffer, and 50-µg samples were analyzed by immunoblotting as shown. GAPDH and Coomassie blue (CB) staining are provided as controls for equal loading. (B) Total skeletal muscle from wild-type (WT) and SSPN-null (SSPN −/− ) mice were solubilized in modified RIPA buffer, and 60 µg of each sample was analyzed by immunoblotting with indicated antibodies. GAPDH is provided as a control for equal loading. (C) Skeletal muscle protein lysates from WT, mdx , utrophin-deficient mdx muscle ( mdx :utr −/− ), and α7 integrin–deficient mdx muscle ( mdx : α7 −/− ) were enriched by sWGA lectin affinity chromatography (sWGA eluate). Immunoblots of 10-µg bound proteins eluted with GlcNAc are shown. Void (unbound) fractions are shown in Fig. S5 B . (D) WT quadriceps were injected with CTX to induce skeletal muscle degeneration/regeneration to evaluate the expression of utrophin and WFA binding without the complications of mdx pathology. Injected muscle cryosections were costained with utrophin (green fluorescence) and embryonic myosin heavy chain (eMHC; red fluorescence) as a marker for newly regenerated fibers 4 d after CTX injection. Serial sections were stained with WFA lectin (green fluorescence). Staining was visualized by indirect immunofluorescence. Actively regenerating myofibers displayed robust utrophin expression and WFA binding around the same nonjunctional areas of the sarcolemma. Mice were 6.5 wk of age at the time of analysis. Bar, 50 μm. Utr, utrophin; Dys, dystrophin; P, phospho; Intg, integrin.

    Article Snippet: sWGA and WFA enrichment of protein lysates 3.5-mg protein samples were incubated with 1.2 ml sWGA-conjugated agarose slurry (AL-1023S; Vector laboratories) or WFA-conjugated agarose slurry (AL-1353; Vector Laboratories) and gently rotated overnight at 4°C. sWGA and WFA agarose was washed four times in RIPA or standard DGC buffer ( ) containing fresh protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mM benzamidine, 0.2 mM PMSF, 5 µM calpain I, and 5 µM calpeptin) to remove unbound proteins.

    Techniques: Activation Assay, Modification, Staining, Mouse Assay, Affinity Chromatography, Western Blot, Injection, Expressing, Binding Assay, Fluorescence, Marker, Immunofluorescence

    Loss of SSPN impairs utrophin expression and glycosylation of α-DG. (A) Skeletal muscle lysates from wild-type (WT) and SSPN-deficient (SSPN −/− ) mice were enriched by sWGA lectin chromatography, and 10-µg protein samples were immunoblotted with the indicated antibodies. (B) Skeletal muscle protein lysates from WT muscle and SSPN-null (SSPN −/− ) mice were enriched by WFA lectin affinity chromatography, and nitrocellulose transfers of 10-µg WFA eluates were probed with the indicated antibodies or incubated with WFA lectin (WFA). Immunoblot exposures for each antibody/lectin staining are identical in Fig. 6 F , permitting direct comparisons. (C) sWGA-enriched protein samples were separated by ultracentrifugation through 5–20% sucrose gradients. Fraction numbers are indicated above the panels, where fraction 1 represents the lightest region of the gradient. Protein samples were analyzed by immunoblotting to the indicated antibodies, and exposures are identical for WT and SSPN-null fractions. (D) Quantitative RT-PCR was used to investigate whether loss of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). Data are expressed relative to that of WT controls. Error bars represent standard deviation ( n = 4 mice per genotype). (E) Skeletal muscle from WT and SSPN-deficient (SSPN −/− ) muscles were solubilized in 60 µg RIPA buffer and analyzed by immunoblots with Galgt2 antibodies. (F) Quantitative RT-PCR was used to investigate the effect of SSPN on utrophin (Utr) mRNA levels. RNA was isolated from WT, SSPN-null (SSPN −/− ), LARGE-null ( myd ), SSPN-deficient myd ( myd :SSPN −/− ), and threefold SSPN-Tg: myd ( myd 3.0 ) skeletal muscle. mRNA expression levels were normalized to GAPDH mRNA. Data are expressed relative to that of WT controls. Error bars represent standard deviation ( n = 3 mice per genotype; ***, P

    Journal: The Journal of Cell Biology

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

    doi: 10.1083/jcb.201110032

    Figure Lengend Snippet: Loss of SSPN impairs utrophin expression and glycosylation of α-DG. (A) Skeletal muscle lysates from wild-type (WT) and SSPN-deficient (SSPN −/− ) mice were enriched by sWGA lectin chromatography, and 10-µg protein samples were immunoblotted with the indicated antibodies. (B) Skeletal muscle protein lysates from WT muscle and SSPN-null (SSPN −/− ) mice were enriched by WFA lectin affinity chromatography, and nitrocellulose transfers of 10-µg WFA eluates were probed with the indicated antibodies or incubated with WFA lectin (WFA). Immunoblot exposures for each antibody/lectin staining are identical in Fig. 6 F , permitting direct comparisons. (C) sWGA-enriched protein samples were separated by ultracentrifugation through 5–20% sucrose gradients. Fraction numbers are indicated above the panels, where fraction 1 represents the lightest region of the gradient. Protein samples were analyzed by immunoblotting to the indicated antibodies, and exposures are identical for WT and SSPN-null fractions. (D) Quantitative RT-PCR was used to investigate whether loss of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). Data are expressed relative to that of WT controls. Error bars represent standard deviation ( n = 4 mice per genotype). (E) Skeletal muscle from WT and SSPN-deficient (SSPN −/− ) muscles were solubilized in 60 µg RIPA buffer and analyzed by immunoblots with Galgt2 antibodies. (F) Quantitative RT-PCR was used to investigate the effect of SSPN on utrophin (Utr) mRNA levels. RNA was isolated from WT, SSPN-null (SSPN −/− ), LARGE-null ( myd ), SSPN-deficient myd ( myd :SSPN −/− ), and threefold SSPN-Tg: myd ( myd 3.0 ) skeletal muscle. mRNA expression levels were normalized to GAPDH mRNA. Data are expressed relative to that of WT controls. Error bars represent standard deviation ( n = 3 mice per genotype; ***, P

    Article Snippet: sWGA and WFA enrichment of protein lysates 3.5-mg protein samples were incubated with 1.2 ml sWGA-conjugated agarose slurry (AL-1023S; Vector laboratories) or WFA-conjugated agarose slurry (AL-1353; Vector Laboratories) and gently rotated overnight at 4°C. sWGA and WFA agarose was washed four times in RIPA or standard DGC buffer ( ) containing fresh protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mM benzamidine, 0.2 mM PMSF, 5 µM calpain I, and 5 µM calpeptin) to remove unbound proteins.

    Techniques: Expressing, Mouse Assay, Chromatography, Affinity Chromatography, Incubation, Staining, Quantitative RT-PCR, Standard Deviation, Western Blot, Isolation

    SSPN increases cell surface glycosylation in mdx muscle. (A and B) Transverse cryosections of quadriceps muscles from SSPN-Tg (A) or Akt transgenic (B) muscles were stained with biotinylated Wisteria floribunda agglutinin (WFA) and visualized by indirect immunofluorescence. Bars, 50 µm. (C) Skeletal muscle protein lysates from the indicated mouse models were enriched by WFA lectin affinity chromatography (WFA enrichment) and analyzed with indicated antibodies and overlayed (O/L) with WFA lectin (WFA O/L). (D) Skeletal muscle protein lysates were enriched by WFA lectin affinity chromatography (WFA enrichment) and subjected to the same analysis as described in C. (E) WFA and laminin overlay assays were performed on protein lysates enriched with succinylated WGA (sWGA) lectin chromatography from mdx and Akt transgenic mdx ( mdx Akt ) muscle. Mice were treated with doxycycline to induce Akt expression in skeletal muscle as described previously ( Peter et al., 2009 ). Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Immunoblotting with antibodies to α-DG is shown. (F) Levels of WFA binding to α-DG were quantitated by densitometry of bands from overlay assays, and data are expressed relative to mdx levels (100%). Error bars represent standard deviation of the mean ( n = 2–3 muscle preps per genotype). (G) Quantitative RT-PCR was used to investigate whether overexpression of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). RNA was isolated from WT, WT 1.5 , mdx , and mdx 1.5 skeletal muscle. Data are expressed relative to non-Tg WT controls. Error bars represent standard error of the mean (*, P

    Journal: The Journal of Cell Biology

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

    doi: 10.1083/jcb.201110032

    Figure Lengend Snippet: SSPN increases cell surface glycosylation in mdx muscle. (A and B) Transverse cryosections of quadriceps muscles from SSPN-Tg (A) or Akt transgenic (B) muscles were stained with biotinylated Wisteria floribunda agglutinin (WFA) and visualized by indirect immunofluorescence. Bars, 50 µm. (C) Skeletal muscle protein lysates from the indicated mouse models were enriched by WFA lectin affinity chromatography (WFA enrichment) and analyzed with indicated antibodies and overlayed (O/L) with WFA lectin (WFA O/L). (D) Skeletal muscle protein lysates were enriched by WFA lectin affinity chromatography (WFA enrichment) and subjected to the same analysis as described in C. (E) WFA and laminin overlay assays were performed on protein lysates enriched with succinylated WGA (sWGA) lectin chromatography from mdx and Akt transgenic mdx ( mdx Akt ) muscle. Mice were treated with doxycycline to induce Akt expression in skeletal muscle as described previously ( Peter et al., 2009 ). Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Immunoblotting with antibodies to α-DG is shown. (F) Levels of WFA binding to α-DG were quantitated by densitometry of bands from overlay assays, and data are expressed relative to mdx levels (100%). Error bars represent standard deviation of the mean ( n = 2–3 muscle preps per genotype). (G) Quantitative RT-PCR was used to investigate whether overexpression of SSPN alters RNA levels of CT GalNAc transferase ( Galgt2 ). RNA was isolated from WT, WT 1.5 , mdx , and mdx 1.5 skeletal muscle. Data are expressed relative to non-Tg WT controls. Error bars represent standard error of the mean (*, P

    Article Snippet: sWGA and WFA enrichment of protein lysates 3.5-mg protein samples were incubated with 1.2 ml sWGA-conjugated agarose slurry (AL-1023S; Vector laboratories) or WFA-conjugated agarose slurry (AL-1353; Vector Laboratories) and gently rotated overnight at 4°C. sWGA and WFA agarose was washed four times in RIPA or standard DGC buffer ( ) containing fresh protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mM benzamidine, 0.2 mM PMSF, 5 µM calpain I, and 5 µM calpeptin) to remove unbound proteins.

    Techniques: Transgenic Assay, Staining, Immunofluorescence, Affinity Chromatography, Whole Genome Amplification, Chromatography, Mouse Assay, Expressing, Laser Capture Microdissection, Binding Assay, Standard Deviation, Quantitative RT-PCR, Over Expression, Isolation

    SSPN facilitates transportation of the UGC to the sarcolemma to restore laminin binding. (A) Transverse cryosections of quadriceps muscles from the indicated mice were stained with antilaminin antibodies. Bar, 50 µm. (B) Skeletal muscles were solubilized in RIPA buffer, and 50 µg of each protein lysate was resolved by SDS-PAGE. Immunoblotting was performed with indicated antibodies. GAPDH and Coomassie blue (CB) loading controls are shown in Fig. S1 . (C and D) sWGA-enriched protein samples were analyzed for laminin binding. Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Blots were also stained with antibodies to α- and β-DG as indicated. (E) Proteins in ER and Golgi membranes (ER/Golgi) were purified from mdx , mdx 1.5 , and mdx 3.0 muscles. Protein samples were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. Staining with endogenous (mouse SSPN [mSSPN]) and exogenous (human SSPN [hSSPN]) is shown. ATF6 and GM130 served as protein markers for the ER and Golgi, respectively. (F) Proteins in ER and Golgi membranes (ER/Golgi) were purified from mdx ( mdx ) and SSPN-deficient mdx ( mdx :SSPN −/− ) muscles and analyzed by immunoblotting. WFA binding to α-DG was assessed using WFA ligand overlay assays (WFA O/L). Lam, laminin; O/L, overlay; Agr, agrin; PItn, plectin-1; Dys, dystrophin; FL, full length; Utr, utrophin; Intg, integrin.

    Journal: The Journal of Cell Biology

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

    doi: 10.1083/jcb.201110032

    Figure Lengend Snippet: SSPN facilitates transportation of the UGC to the sarcolemma to restore laminin binding. (A) Transverse cryosections of quadriceps muscles from the indicated mice were stained with antilaminin antibodies. Bar, 50 µm. (B) Skeletal muscles were solubilized in RIPA buffer, and 50 µg of each protein lysate was resolved by SDS-PAGE. Immunoblotting was performed with indicated antibodies. GAPDH and Coomassie blue (CB) loading controls are shown in Fig. S1 . (C and D) sWGA-enriched protein samples were analyzed for laminin binding. Laminin overlays (Lam O/L) represent binding to immobilized α-DG on nitrocellulose transfers. Blots were also stained with antibodies to α- and β-DG as indicated. (E) Proteins in ER and Golgi membranes (ER/Golgi) were purified from mdx , mdx 1.5 , and mdx 3.0 muscles. Protein samples were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. Staining with endogenous (mouse SSPN [mSSPN]) and exogenous (human SSPN [hSSPN]) is shown. ATF6 and GM130 served as protein markers for the ER and Golgi, respectively. (F) Proteins in ER and Golgi membranes (ER/Golgi) were purified from mdx ( mdx ) and SSPN-deficient mdx ( mdx :SSPN −/− ) muscles and analyzed by immunoblotting. WFA binding to α-DG was assessed using WFA ligand overlay assays (WFA O/L). Lam, laminin; O/L, overlay; Agr, agrin; PItn, plectin-1; Dys, dystrophin; FL, full length; Utr, utrophin; Intg, integrin.

    Article Snippet: sWGA and WFA enrichment of protein lysates 3.5-mg protein samples were incubated with 1.2 ml sWGA-conjugated agarose slurry (AL-1023S; Vector laboratories) or WFA-conjugated agarose slurry (AL-1353; Vector Laboratories) and gently rotated overnight at 4°C. sWGA and WFA agarose was washed four times in RIPA or standard DGC buffer ( ) containing fresh protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mM benzamidine, 0.2 mM PMSF, 5 µM calpain I, and 5 µM calpeptin) to remove unbound proteins.

    Techniques: Binding Assay, Mouse Assay, Staining, SDS Page, Laser Capture Microdissection, Purification

    SSPN regulates utrophin levels and glycosylation of α-DG in myd mice. (A) Transverse cryosections of quadriceps muscles were stained with the indicated antibodies and overlayed with biotinylated WFA. (B) Transverse cryosections of quadriceps muscles were stained with indicated antibodies and overlayed with biotinylated WFA. Staining with IIH6 antibodies, which recognize LARGE epitopes on α-DG, was not detected in myd samples, as expected ( Fig. S4 ). Transverse cryosections of skeletal muscle from 4–6-wk-old myd , SSPN-Tg: myd ( myd 3.0 ), and SSPN-deficient myd ( myd :SSPN −/− ) mice were stained with H E. Muscle sections were stained with antibodies to embryonic myosin heavy chain (eMHC; green) as a marker for newly regenerated myofibers. Mice were injected with Evans blue dye (EBD), a marker for membrane instability (visualized by red fluorescence). Sections were costained with laminin antibodies (green fluorescence) to visualize individual fibers. (C) Quantification of central nucleation, Evans blue dye–positive fibers, and eMHC-positive fibers is expressed as a percentage of total fibers. Error bars represent standard deviation of the mean ( n = 4 quadriceps per genotype). (D–G) Skeletal muscles from the indicated mice were enriched using either sWGA or WFA lectin chromatography. Immunoblots of 10-µg protein eluates are shown. A.U., arbitrary unit; Utr, utrophin. Bars, 50 µm.

    Journal: The Journal of Cell Biology

    Article Title: Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration

    doi: 10.1083/jcb.201110032

    Figure Lengend Snippet: SSPN regulates utrophin levels and glycosylation of α-DG in myd mice. (A) Transverse cryosections of quadriceps muscles were stained with the indicated antibodies and overlayed with biotinylated WFA. (B) Transverse cryosections of quadriceps muscles were stained with indicated antibodies and overlayed with biotinylated WFA. Staining with IIH6 antibodies, which recognize LARGE epitopes on α-DG, was not detected in myd samples, as expected ( Fig. S4 ). Transverse cryosections of skeletal muscle from 4–6-wk-old myd , SSPN-Tg: myd ( myd 3.0 ), and SSPN-deficient myd ( myd :SSPN −/− ) mice were stained with H E. Muscle sections were stained with antibodies to embryonic myosin heavy chain (eMHC; green) as a marker for newly regenerated myofibers. Mice were injected with Evans blue dye (EBD), a marker for membrane instability (visualized by red fluorescence). Sections were costained with laminin antibodies (green fluorescence) to visualize individual fibers. (C) Quantification of central nucleation, Evans blue dye–positive fibers, and eMHC-positive fibers is expressed as a percentage of total fibers. Error bars represent standard deviation of the mean ( n = 4 quadriceps per genotype). (D–G) Skeletal muscles from the indicated mice were enriched using either sWGA or WFA lectin chromatography. Immunoblots of 10-µg protein eluates are shown. A.U., arbitrary unit; Utr, utrophin. Bars, 50 µm.

    Article Snippet: sWGA and WFA enrichment of protein lysates 3.5-mg protein samples were incubated with 1.2 ml sWGA-conjugated agarose slurry (AL-1023S; Vector laboratories) or WFA-conjugated agarose slurry (AL-1353; Vector Laboratories) and gently rotated overnight at 4°C. sWGA and WFA agarose was washed four times in RIPA or standard DGC buffer ( ) containing fresh protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mM benzamidine, 0.2 mM PMSF, 5 µM calpain I, and 5 µM calpeptin) to remove unbound proteins.

    Techniques: Mouse Assay, Staining, Marker, Injection, Fluorescence, Standard Deviation, Chromatography, Western Blot

    The matrix polysaccharide Pel was stained and quantified in P. aeruginosa strain PA14 biofilms using florescence marker bound to Pel-specific lectin in M63 media plus arginine, a biofilm assay media 97 ) and ASMi medium (p

    Journal: bioRxiv

    Article Title: Pseudomonas aeruginosa and Candida albicans both accumulate greater biomass in dual species biofilms under flow

    doi: 10.1101/2020.10.29.361139

    Figure Lengend Snippet: The matrix polysaccharide Pel was stained and quantified in P. aeruginosa strain PA14 biofilms using florescence marker bound to Pel-specific lectin in M63 media plus arginine, a biofilm assay media 97 ) and ASMi medium (p

    Article Snippet: A 458-nm laser line was used to excite Wisteria floribunda lectin stain in the case of Pel quantification experiments.

    Techniques: Staining, Marker, Biofilm Production Assay

    Chondroitinase treatment of brain sections prior to binding by WFA . Binding of Wisteria floribunda agglutinin (WFA) lectin after treatment with Chase. Paraformaldehyde-fixed coronal sections from (A and C) rat and (B and D) mouse brain were probed with biotinylated-WFA (A and B) before and (C and D) after Chase treatment. Arrow = retrosplenial cortex; arrow head = parietal cortex. Note the near elimination of reactivity after Chase treatment. All images were captured at 25× magnification. Scale bar represents 100 μm.

    Journal: BMC Neuroscience

    Article Title: Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain

    doi: 10.1186/1471-2202-9-14

    Figure Lengend Snippet: Chondroitinase treatment of brain sections prior to binding by WFA . Binding of Wisteria floribunda agglutinin (WFA) lectin after treatment with Chase. Paraformaldehyde-fixed coronal sections from (A and C) rat and (B and D) mouse brain were probed with biotinylated-WFA (A and B) before and (C and D) after Chase treatment. Arrow = retrosplenial cortex; arrow head = parietal cortex. Note the near elimination of reactivity after Chase treatment. All images were captured at 25× magnification. Scale bar represents 100 μm.

    Article Snippet: For WFA blotting, the membranes were washed with Buffer B for 5 minutes, blocked in 1% bovine serum albumin diluted in Buffer B for 1 hour and probed for 2 hours using biotinylated Wisteria floribunda lectin (1:10,000 in 1% BSA, Vector Laboratories, Burlingame, CA) as the primary binding reagent.

    Techniques: Binding Assay