pha e  (Vector Laboratories)


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    Structured Review

    Vector Laboratories pha e
    Lectin histochemical staining profiles in sections of OA cartilage. (a) Binding of PNA to complex chondrons of a severely degenerated cartilage region could be completely blocked with lactose (inset) ascertaining carbohydrate-specific binding. (b) Omission of the incubation step with biotinylated LEA (first-step reagent) from processing excluded probe-independent signal generation. (c, d) ConA staining: staining pattern of MS ≤4 regions included chondrocytes in deep zones of cartilage (c) . Intense staining of matrix and chondrons (inset) in MS ≥9 regions (d) . (e) PSA staining: positivity of chondrons (inset) and matrix, predominantly in MS ≥9 cartilage. (f, g) <t>PHA-E</t> staining: whereas MS ≤4 regions were negative (f) , MS ≥9 areas (g) presented positive chondrons (inset) and matrix. (h) PHA-L staining: binding sites were restricted to chondrons (insert) and matrix of MS ≥9 cartilage. (i-j) VAA staining: whereas the chondrons of MS ≤4 areas were negative (i) , reactivity was observed both in chondrons (inset) and matrix of MS ≥9 cartilage (j) . (k) LEA staining: reactivity for chondrons (inset) and matrix of MS ≥9 regions. (l) MAA-I staining: reactivity included chondrons (inset) and matrix of MS ≥9 cartilage. (m, n) SNA staining: weak staining of matrix and no staining of chondrons in MS ≤4 cartilage (m) , whereas both chondrons (inset) and matrix were positive in MS ≥9 regions (n) . (o) <t>DBA</t> staining: positivity in chondrons (inset) and matrix of MS ≥9 cartilage. (p, q) PNA staining: positive chondrocytes sparely distributed in the deeper zones of MS ≤4 cartilage ( p ; arrows, inset). In MS ≥9 cartilage (q) , intense matrix staining was observed, whereas chondrons were mostly negative (inset). (r) JAC staining: absent in chondrons (inset), but present in superficial zones of MS ≥9 cartilage. Bars in inserts of d,e,g,l,n,p,q,r: 50μm. Bars in inserts of h , j , k , o : 100μm. MS, Mankin score; OA, osteoarthritis.
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    Images

    1) Product Images from "Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein"

    Article Title: Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar4330

    Lectin histochemical staining profiles in sections of OA cartilage. (a) Binding of PNA to complex chondrons of a severely degenerated cartilage region could be completely blocked with lactose (inset) ascertaining carbohydrate-specific binding. (b) Omission of the incubation step with biotinylated LEA (first-step reagent) from processing excluded probe-independent signal generation. (c, d) ConA staining: staining pattern of MS ≤4 regions included chondrocytes in deep zones of cartilage (c) . Intense staining of matrix and chondrons (inset) in MS ≥9 regions (d) . (e) PSA staining: positivity of chondrons (inset) and matrix, predominantly in MS ≥9 cartilage. (f, g) PHA-E staining: whereas MS ≤4 regions were negative (f) , MS ≥9 areas (g) presented positive chondrons (inset) and matrix. (h) PHA-L staining: binding sites were restricted to chondrons (insert) and matrix of MS ≥9 cartilage. (i-j) VAA staining: whereas the chondrons of MS ≤4 areas were negative (i) , reactivity was observed both in chondrons (inset) and matrix of MS ≥9 cartilage (j) . (k) LEA staining: reactivity for chondrons (inset) and matrix of MS ≥9 regions. (l) MAA-I staining: reactivity included chondrons (inset) and matrix of MS ≥9 cartilage. (m, n) SNA staining: weak staining of matrix and no staining of chondrons in MS ≤4 cartilage (m) , whereas both chondrons (inset) and matrix were positive in MS ≥9 regions (n) . (o) DBA staining: positivity in chondrons (inset) and matrix of MS ≥9 cartilage. (p, q) PNA staining: positive chondrocytes sparely distributed in the deeper zones of MS ≤4 cartilage ( p ; arrows, inset). In MS ≥9 cartilage (q) , intense matrix staining was observed, whereas chondrons were mostly negative (inset). (r) JAC staining: absent in chondrons (inset), but present in superficial zones of MS ≥9 cartilage. Bars in inserts of d,e,g,l,n,p,q,r: 50μm. Bars in inserts of h , j , k , o : 100μm. MS, Mankin score; OA, osteoarthritis.
    Figure Legend Snippet: Lectin histochemical staining profiles in sections of OA cartilage. (a) Binding of PNA to complex chondrons of a severely degenerated cartilage region could be completely blocked with lactose (inset) ascertaining carbohydrate-specific binding. (b) Omission of the incubation step with biotinylated LEA (first-step reagent) from processing excluded probe-independent signal generation. (c, d) ConA staining: staining pattern of MS ≤4 regions included chondrocytes in deep zones of cartilage (c) . Intense staining of matrix and chondrons (inset) in MS ≥9 regions (d) . (e) PSA staining: positivity of chondrons (inset) and matrix, predominantly in MS ≥9 cartilage. (f, g) PHA-E staining: whereas MS ≤4 regions were negative (f) , MS ≥9 areas (g) presented positive chondrons (inset) and matrix. (h) PHA-L staining: binding sites were restricted to chondrons (insert) and matrix of MS ≥9 cartilage. (i-j) VAA staining: whereas the chondrons of MS ≤4 areas were negative (i) , reactivity was observed both in chondrons (inset) and matrix of MS ≥9 cartilage (j) . (k) LEA staining: reactivity for chondrons (inset) and matrix of MS ≥9 regions. (l) MAA-I staining: reactivity included chondrons (inset) and matrix of MS ≥9 cartilage. (m, n) SNA staining: weak staining of matrix and no staining of chondrons in MS ≤4 cartilage (m) , whereas both chondrons (inset) and matrix were positive in MS ≥9 regions (n) . (o) DBA staining: positivity in chondrons (inset) and matrix of MS ≥9 cartilage. (p, q) PNA staining: positive chondrocytes sparely distributed in the deeper zones of MS ≤4 cartilage ( p ; arrows, inset). In MS ≥9 cartilage (q) , intense matrix staining was observed, whereas chondrons were mostly negative (inset). (r) JAC staining: absent in chondrons (inset), but present in superficial zones of MS ≥9 cartilage. Bars in inserts of d,e,g,l,n,p,q,r: 50μm. Bars in inserts of h , j , k , o : 100μm. MS, Mankin score; OA, osteoarthritis.

    Techniques Used: Staining, Binding Assay, Incubation, Mass Spectrometry

    2) Product Images from "Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies"

    Article Title: Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies

    Journal: Scientific Reports

    doi: 10.1038/s41598-022-16605-5

    Lectin and Western blot analyses of total glycoproteins in MDCK, SIAT1, and hCK cells. Total cell lysates of MDCK, SIAT1, and hCK cell lines were treated with PNGase F (PNGaseF), Neuraminidase A (NeuA), or Neuraminidase S (NeuS), and separated by SDS-PAGE. Total extracts of cells at passage 3 (top) and passage 23 (bottom) ( A–F ), plus PNGaseF/neuraminidases mixture (Enzyme mix) control were loaded on the gels. ( A ) Gel stained with Coomassie Brilliant Blue solution. ( B ) Transferred total glycoproteins analyzed by lectin blot using ConA; ( C ) SNA; ( D ) MAL-I; and ( E ) PHA-E. ( F ) Total glycoproteins from cells at passage 3, and 23 were Western blotted with the antibody O6.
    Figure Legend Snippet: Lectin and Western blot analyses of total glycoproteins in MDCK, SIAT1, and hCK cells. Total cell lysates of MDCK, SIAT1, and hCK cell lines were treated with PNGase F (PNGaseF), Neuraminidase A (NeuA), or Neuraminidase S (NeuS), and separated by SDS-PAGE. Total extracts of cells at passage 3 (top) and passage 23 (bottom) ( A–F ), plus PNGaseF/neuraminidases mixture (Enzyme mix) control were loaded on the gels. ( A ) Gel stained with Coomassie Brilliant Blue solution. ( B ) Transferred total glycoproteins analyzed by lectin blot using ConA; ( C ) SNA; ( D ) MAL-I; and ( E ) PHA-E. ( F ) Total glycoproteins from cells at passage 3, and 23 were Western blotted with the antibody O6.

    Techniques Used: Western Blot, SDS Page, Staining

    3) Product Images from "Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology"

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

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-014-0101-0

    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 1 .
    Figure Legend 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 1 .

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

    4) Product Images from "DNA damage is overcome by TRIP13 overexpression during cisplatin nephrotoxicity"

    Article Title: DNA damage is overcome by TRIP13 overexpression during cisplatin nephrotoxicity

    Journal: JCI Insight

    doi: 10.1172/jci.insight.139092

    Genomic targeting construct of Trip13 overexpression. ( A ) Targeting construct was designed for integration into the ROSA26 region, and the expression cassette was designed with the CAG promoter-floxed stop codon-Trip13 cDNA-T2A-EGFP-polyA. 2A, protease cleavage site ( Trip13 cDNA was FLAG tagged at the 3′ end); CAG, CMV-IE enhancer/chicken β-actin/rabbit β-globin. ( B ) Genomic DNA isolated from mouse pups was PCR analyzed using specific primers to differentiate wild-type, floxed stop, and Cre-containing mice. Wild-type (WT) = 453 bp; floxed stop (flox) = 616 bp; and Cre = 400 bp. M = 1 kb ladder; lanes 1, 4, and 7 = F1/R1 primers (wild-type); 2, 5, and 8 = F2/R1 primers (floxed Trip13); and 3, 6, and 9 = GGT1-Cre primers (Cre); Trip13 st/st , Trip13 Stop/Stop . ( C ) Western blot analysis of FLAG-tagged TRIP13 and GFP protein expression in harvested tissues from Trip13 ΔStop mice. GAPDH was shown as a loading control. K, kidney; Duo, duodenum; Hrt, heart; Br, brain; Lu, lungs; Spl, spleen. ( D – H ) Immunofluorescence was performed on FFPE Trip13 Stop/Stop and Trip13 ΔStop kidneys to detect GFP expression. GFP was detected by Alexa Fluor 555 fluorescence (red color) in ( G ) Trip13 Stop/Stop and ( D , F , and H ) Trip13 ΔStop kidneys. E shows a negative control kidney section incubated without primary GFP antibody (no primary). To determine proximal tubules ( F and G ) or collecting ducts ( H ), Alexa Fluor 488 (green) fluorescence was detected using PVA-E (brush border of proximal tubules) or DBA (collecting ducts). Nuclei were stained with DAPI (blue). Dashed lines in F indicate PVA-E–positive tubules with minimal to undetectable expression of GFP, showing mosaicism of Cre expression. ( I and J ) Immunohistochemical staining for GFP in cisplatin-treated Trip13 ΔStop and Trip13 Stop/Stop mouse lungs. All DAB-stained sections were counterstained with hematoxylin. Scale bar: 200 μm ( D , E , I , and J ), 100 μm ( F – H ). PVA-E, Phaseolus vulgaris erthyroagglutinin; DBA, Dolicus biflorus agglutinin.
    Figure Legend Snippet: Genomic targeting construct of Trip13 overexpression. ( A ) Targeting construct was designed for integration into the ROSA26 region, and the expression cassette was designed with the CAG promoter-floxed stop codon-Trip13 cDNA-T2A-EGFP-polyA. 2A, protease cleavage site ( Trip13 cDNA was FLAG tagged at the 3′ end); CAG, CMV-IE enhancer/chicken β-actin/rabbit β-globin. ( B ) Genomic DNA isolated from mouse pups was PCR analyzed using specific primers to differentiate wild-type, floxed stop, and Cre-containing mice. Wild-type (WT) = 453 bp; floxed stop (flox) = 616 bp; and Cre = 400 bp. M = 1 kb ladder; lanes 1, 4, and 7 = F1/R1 primers (wild-type); 2, 5, and 8 = F2/R1 primers (floxed Trip13); and 3, 6, and 9 = GGT1-Cre primers (Cre); Trip13 st/st , Trip13 Stop/Stop . ( C ) Western blot analysis of FLAG-tagged TRIP13 and GFP protein expression in harvested tissues from Trip13 ΔStop mice. GAPDH was shown as a loading control. K, kidney; Duo, duodenum; Hrt, heart; Br, brain; Lu, lungs; Spl, spleen. ( D – H ) Immunofluorescence was performed on FFPE Trip13 Stop/Stop and Trip13 ΔStop kidneys to detect GFP expression. GFP was detected by Alexa Fluor 555 fluorescence (red color) in ( G ) Trip13 Stop/Stop and ( D , F , and H ) Trip13 ΔStop kidneys. E shows a negative control kidney section incubated without primary GFP antibody (no primary). To determine proximal tubules ( F and G ) or collecting ducts ( H ), Alexa Fluor 488 (green) fluorescence was detected using PVA-E (brush border of proximal tubules) or DBA (collecting ducts). Nuclei were stained with DAPI (blue). Dashed lines in F indicate PVA-E–positive tubules with minimal to undetectable expression of GFP, showing mosaicism of Cre expression. ( I and J ) Immunohistochemical staining for GFP in cisplatin-treated Trip13 ΔStop and Trip13 Stop/Stop mouse lungs. All DAB-stained sections were counterstained with hematoxylin. Scale bar: 200 μm ( D , E , I , and J ), 100 μm ( F – H ). PVA-E, Phaseolus vulgaris erthyroagglutinin; DBA, Dolicus biflorus agglutinin.

    Techniques Used: Construct, Over Expression, Expressing, Isolation, Polymerase Chain Reaction, Mouse Assay, Western Blot, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Fluorescence, Negative Control, Incubation, Staining, Immunohistochemistry

    5) Product Images from "Shedding of N-acetylglucosaminyltransferase-V is regulated by maturity of cellular N-glycan"

    Article Title: Shedding of N-acetylglucosaminyltransferase-V is regulated by maturity of cellular N-glycan

    Journal: Communications Biology

    doi: 10.1038/s42003-022-03697-y

    Increased protein level and activity of GnT-V in complex-type N -glycan-deficient cells. a Scheme of biosynthesis pathway of N -glycans. Glycan symbols are according to the standard symbol nomenclature for glycans 93 . Asn: asparagine. b Lectin blotting of proteins from HEK293 cells treated with or without kifunensine (kif). c In vitro enzyme assays of GnTs (left) and FUT8 (right) in HEK293 cells treated with or without kifunensine. For GnTs, GnGnbi-PA was used as an acceptor substrate. For FUT8, GnGnbi-Asn-PNS was used as an acceptor substrate. The product peak for each enzyme reaction was identified from control reactions using each purified recombinant enzyme, as shown in Supplementary Fig. 1 . d Quantification of the activities of GnTs and FUT8 as measured in ( c ). Error bars represent SD ( n = 4). Statistical analysis was by unpaired Student’s t -test. e Proteins from HEK293 cells treated with or without kifunensine were blotted for GnT-V and GAPDH. Before SDS-PAGE, cell lysates were treated with or without PNGase F (PNG-F). The graph shows quantification of the GnT-V signals in western blots. Error bars represent SD ( n = 3). Statistical analysis was by Welch’s t -test. f Proteins from wild-type (WT), GnT-I-deficient (I-KO), and GnT-I (myc-tagged)-rescued HEK293 cells were blotted with Con A, PHA-L4, or anti-myc antibody. g Proteins from WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells were treated with or without PNGase F and blotted for GnT-V and GAPDH. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 3). Statistical analysis was by one-way analysis of variance (ANOVA) with post-hoc Tukey test. h The cellular activity of GnT-V in WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. i Localization of Halo-tagged GnT-V (V-Halo) visualized in B16 cells treated with or without kifunensine. (Upper) GM130 and Golgin97 were stained as cis - and trans -Golgi markers, respectively. (Lower) Line plots of relative fluorescence intensity are shown. Arrows indicate the plotted regions. Scale bars: 10 or 2 µm. Staining with Calnexin, an ER marker, was shown in Supplementary Fig. 5 . * p
    Figure Legend Snippet: Increased protein level and activity of GnT-V in complex-type N -glycan-deficient cells. a Scheme of biosynthesis pathway of N -glycans. Glycan symbols are according to the standard symbol nomenclature for glycans 93 . Asn: asparagine. b Lectin blotting of proteins from HEK293 cells treated with or without kifunensine (kif). c In vitro enzyme assays of GnTs (left) and FUT8 (right) in HEK293 cells treated with or without kifunensine. For GnTs, GnGnbi-PA was used as an acceptor substrate. For FUT8, GnGnbi-Asn-PNS was used as an acceptor substrate. The product peak for each enzyme reaction was identified from control reactions using each purified recombinant enzyme, as shown in Supplementary Fig. 1 . d Quantification of the activities of GnTs and FUT8 as measured in ( c ). Error bars represent SD ( n = 4). Statistical analysis was by unpaired Student’s t -test. e Proteins from HEK293 cells treated with or without kifunensine were blotted for GnT-V and GAPDH. Before SDS-PAGE, cell lysates were treated with or without PNGase F (PNG-F). The graph shows quantification of the GnT-V signals in western blots. Error bars represent SD ( n = 3). Statistical analysis was by Welch’s t -test. f Proteins from wild-type (WT), GnT-I-deficient (I-KO), and GnT-I (myc-tagged)-rescued HEK293 cells were blotted with Con A, PHA-L4, or anti-myc antibody. g Proteins from WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells were treated with or without PNGase F and blotted for GnT-V and GAPDH. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 3). Statistical analysis was by one-way analysis of variance (ANOVA) with post-hoc Tukey test. h The cellular activity of GnT-V in WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. i Localization of Halo-tagged GnT-V (V-Halo) visualized in B16 cells treated with or without kifunensine. (Upper) GM130 and Golgin97 were stained as cis - and trans -Golgi markers, respectively. (Lower) Line plots of relative fluorescence intensity are shown. Arrows indicate the plotted regions. Scale bars: 10 or 2 µm. Staining with Calnexin, an ER marker, was shown in Supplementary Fig. 5 . * p

    Techniques Used: Activity Assay, In Vitro, Purification, Recombinant, SDS Page, Western Blot, Staining, Fluorescence, Marker

    Inhibition of SPPL3-dependent secretion of GnT-V in complex-type N -glycan-deficient cells. a Relative mRNA expression of MGAT5 (encoding GnT-V) in WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells. The levels of MGAT5 mRNA were normalized by those of rRNA. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. b Proteins from HEK293 cells treated with dimethylsulfoxide (DMSO), MG132, or chloroquine (CQ) were blotted for GnT-V, β-catenin, p62, or GAPDH. The samples were treated with PNGase F (PNG-F) before SDS-PAGE. c The activity of GnT-V in DMSO-, MG132-, or CQ-treated HEK293 cells. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Dunnett test. d Proteins in the lysates of WT and GnT-I-deficient cells and in the culture media of these cells were blotted for GnT-V or GAPDH. e WT and SPPL3-deficient B16 cells were treated with or without kifunensine (kif). Proteins in the cell lysates and the culture media of these cells were blotted for GnT-V, APP, or GAPDH. The samples were treated with or without PNGase F before SDS-PAGE. APP was blotted as a positive control of secreted protein 94 . The proteins in the cell lysates were also blotted with Con A or PHA-L4. f The cellular activity of GnT-V in WT and SPPL3-KO B16 cells treated with or without kifunensine. Error bars represent SD ( n = 3). Statistical analysis was by two-way ANOVA with post-hoc Sidak test. *** p
    Figure Legend Snippet: Inhibition of SPPL3-dependent secretion of GnT-V in complex-type N -glycan-deficient cells. a Relative mRNA expression of MGAT5 (encoding GnT-V) in WT, GnT-I-deficient (I-KO), and GnT-I-rescued HEK293 cells. The levels of MGAT5 mRNA were normalized by those of rRNA. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. b Proteins from HEK293 cells treated with dimethylsulfoxide (DMSO), MG132, or chloroquine (CQ) were blotted for GnT-V, β-catenin, p62, or GAPDH. The samples were treated with PNGase F (PNG-F) before SDS-PAGE. c The activity of GnT-V in DMSO-, MG132-, or CQ-treated HEK293 cells. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Dunnett test. d Proteins in the lysates of WT and GnT-I-deficient cells and in the culture media of these cells were blotted for GnT-V or GAPDH. e WT and SPPL3-deficient B16 cells were treated with or without kifunensine (kif). Proteins in the cell lysates and the culture media of these cells were blotted for GnT-V, APP, or GAPDH. The samples were treated with or without PNGase F before SDS-PAGE. APP was blotted as a positive control of secreted protein 94 . The proteins in the cell lysates were also blotted with Con A or PHA-L4. f The cellular activity of GnT-V in WT and SPPL3-KO B16 cells treated with or without kifunensine. Error bars represent SD ( n = 3). Statistical analysis was by two-way ANOVA with post-hoc Sidak test. *** p

    Techniques Used: Inhibition, Expressing, SDS Page, Activity Assay, Positive Control

    Requirement of glycan terminal modifications for efficient GnT-V shedding. a Proteins from WT and SLC35C1-KO (C1-KO) HEK293 cells were blotted with AAL or anti-actin antibody. b The activity of GnT-V in WT and SLC35C1-KO HEK293 cells. Error bars represent SD ( n = 4). Statistical analysis was by unpaired Student’s t -test. c C -terminally 3 × FLAG-tagged GnT-V WT was expressed in WT and SLC35C1-KO HEK293 cells, and secretion of GnT-V in these cells were assessed by quantify the signal ratio of secreted GnT-V (in medium) to cellular GnT-V. Error bars represent SD ( n = 3). Statistical analysis was by unpaired Student’s t -test. d Proteins from WT, SLC35A2-KO (A2-KO), and SLC35A2-rescued HEK293 cells treated with or without PNGase F were blotted with RCA-I, and anti-GnT-V and anti-GAPDH antibodies. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 5). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. e The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. f Proteins from WT, SLC35A1-KO (A1-KO), and SLC35A1-rescued HEK293 cells treated with or without PNGase F were blotted with RCA-I, and anti-GnT-V and anti-GAPDH antibodies. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. g The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. h Cellular and secreted GnT-V activity from WT, A1-KO and A2-KO HEK293 cells were examined in vitro. The graph shows the relative activity of the ratio of secreted GnT-V (in medium) to cellular GnT-V. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Dunnett test. i Proteins from WT, GnT-III-KO (III-KO), and GnT-III (myc-tagged)-rescued HEK293 cells treated with or without PNGase F were blotted with PHA-E4, and anti-myc, anti-GnT-V, and anti-GAPDH antibodies. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. j The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. k Proteins from WT, GnT-IVa and IVb-DKO (IVa,b-DKO), and GnT-IVa (myc-tagged)-rescued HEK293 cells were blotted with DSA and anti-myc antibody. l The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. m GnT-V WT or its catalytically inactive E297A mutant was expressed in GnT-V-KO (V-KO) HEK293 cells. Proteins in the cell lysates and the culture media of these cells were blotted with PHA-L4, and anti-GnT-V, anti-APP, anti-ZsGreen1, or anti-actin antibodies. The graph shows quantification of the signal ratio of secreted GnT-V to cellular GnT-V. APP was blotted as a positive control of secreted protein. Error bars represent SD ( n = 3). Statistical analysis was by Welch’s t -test. * p
    Figure Legend Snippet: Requirement of glycan terminal modifications for efficient GnT-V shedding. a Proteins from WT and SLC35C1-KO (C1-KO) HEK293 cells were blotted with AAL or anti-actin antibody. b The activity of GnT-V in WT and SLC35C1-KO HEK293 cells. Error bars represent SD ( n = 4). Statistical analysis was by unpaired Student’s t -test. c C -terminally 3 × FLAG-tagged GnT-V WT was expressed in WT and SLC35C1-KO HEK293 cells, and secretion of GnT-V in these cells were assessed by quantify the signal ratio of secreted GnT-V (in medium) to cellular GnT-V. Error bars represent SD ( n = 3). Statistical analysis was by unpaired Student’s t -test. d Proteins from WT, SLC35A2-KO (A2-KO), and SLC35A2-rescued HEK293 cells treated with or without PNGase F were blotted with RCA-I, and anti-GnT-V and anti-GAPDH antibodies. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 5). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. e The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. f Proteins from WT, SLC35A1-KO (A1-KO), and SLC35A1-rescued HEK293 cells treated with or without PNGase F were blotted with RCA-I, and anti-GnT-V and anti-GAPDH antibodies. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. g The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. h Cellular and secreted GnT-V activity from WT, A1-KO and A2-KO HEK293 cells were examined in vitro. The graph shows the relative activity of the ratio of secreted GnT-V (in medium) to cellular GnT-V. Error bars represent SD ( n = 3). Statistical analysis was by one-way ANOVA with post-hoc Dunnett test. i Proteins from WT, GnT-III-KO (III-KO), and GnT-III (myc-tagged)-rescued HEK293 cells treated with or without PNGase F were blotted with PHA-E4, and anti-myc, anti-GnT-V, and anti-GAPDH antibodies. The graph shows quantification of the GnT-V signals in PNGase F-treated blots. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. j The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. k Proteins from WT, GnT-IVa and IVb-DKO (IVa,b-DKO), and GnT-IVa (myc-tagged)-rescued HEK293 cells were blotted with DSA and anti-myc antibody. l The cellular activity of GnT-V. Error bars represent SD ( n = 4). Statistical analysis was by one-way ANOVA with post-hoc Tukey test. m GnT-V WT or its catalytically inactive E297A mutant was expressed in GnT-V-KO (V-KO) HEK293 cells. Proteins in the cell lysates and the culture media of these cells were blotted with PHA-L4, and anti-GnT-V, anti-APP, anti-ZsGreen1, or anti-actin antibodies. The graph shows quantification of the signal ratio of secreted GnT-V to cellular GnT-V. APP was blotted as a positive control of secreted protein. Error bars represent SD ( n = 3). Statistical analysis was by Welch’s t -test. * p

    Techniques Used: Activity Assay, In Vitro, Mutagenesis, Positive Control

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  • 86
    Vector Laboratories pha e
    Lectin histochemical staining profiles in sections of OA cartilage. (a) Binding of PNA to complex chondrons of a severely degenerated cartilage region could be completely blocked with lactose (inset) ascertaining carbohydrate-specific binding. (b) Omission of the incubation step with biotinylated LEA (first-step reagent) from processing excluded probe-independent signal generation. (c, d) ConA staining: staining pattern of MS ≤4 regions included chondrocytes in deep zones of cartilage (c) . Intense staining of matrix and chondrons (inset) in MS ≥9 regions (d) . (e) PSA staining: positivity of chondrons (inset) and matrix, predominantly in MS ≥9 cartilage. (f, g) <t>PHA-E</t> staining: whereas MS ≤4 regions were negative (f) , MS ≥9 areas (g) presented positive chondrons (inset) and matrix. (h) PHA-L staining: binding sites were restricted to chondrons (insert) and matrix of MS ≥9 cartilage. (i-j) VAA staining: whereas the chondrons of MS ≤4 areas were negative (i) , reactivity was observed both in chondrons (inset) and matrix of MS ≥9 cartilage (j) . (k) LEA staining: reactivity for chondrons (inset) and matrix of MS ≥9 regions. (l) MAA-I staining: reactivity included chondrons (inset) and matrix of MS ≥9 cartilage. (m, n) SNA staining: weak staining of matrix and no staining of chondrons in MS ≤4 cartilage (m) , whereas both chondrons (inset) and matrix were positive in MS ≥9 regions (n) . (o) <t>DBA</t> staining: positivity in chondrons (inset) and matrix of MS ≥9 cartilage. (p, q) PNA staining: positive chondrocytes sparely distributed in the deeper zones of MS ≤4 cartilage ( p ; arrows, inset). In MS ≥9 cartilage (q) , intense matrix staining was observed, whereas chondrons were mostly negative (inset). (r) JAC staining: absent in chondrons (inset), but present in superficial zones of MS ≥9 cartilage. Bars in inserts of d,e,g,l,n,p,q,r: 50μm. Bars in inserts of h , j , k , o : 100μm. MS, Mankin score; OA, osteoarthritis.
    Pha E, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Vector Laboratories rabbit anti pha l antibody
    Electron micrographs of a <t>PHA-L+</t> type 1 terminal (T1t) making synaptic contact (arrowhead) with the base of a proximal dendrite (den) emerging from an unlabeled putative pyramidal cell perikaryon (Pyr, pk). Scale bar: 0.5 µm.
    Rabbit Anti Pha L Antibody, 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
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    Vector Laboratories e pha
    (A) Schematic flow of glycoproteomic experiments. Membrane proteins were analyzed by nanoflow-ESI–RPLC–MS/MS before <t>E-PHA</t> capture to verify equivalent protein quality and quantity. <t>Biotinylated</t> E-PHA was added to membrane extracted proteins, and glycoproteins with bisecting glycans were captured using streptavidin magnetic resin prior to nanoflow-ESI–RPLC–MS/MS. Membrane glycoproteins enriched in tumor tissues relative to nonmalignant tissues by E-PHA capture by at least 1.5 times or 150% by spectral counts were identified and are listed in Table 1 . (B) Venn diagram showing the percentage of glycoproteins in Table 1 from endometrioid only (13% purple), serous only (6% blue), or shared (81% blue/purple). (C) Functional annotation of E-PHA enriched membrane glycoproteins using DAVID 2009.
    E Pha, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Laboratories pha e agarose
    Identification of integrin β1 as the target protein bearing bisecting GlcNAc. (a) Intact glycoproteomic analysis by combination <t>of</t> <t>PHA‐E</t> enrichment and LC‐MS/MS (schematic). (b) Venn diagram of numbers of identified glycoproteins bearing bisecting GlcNAc structures in control and MDA‐231/MGAT3. (c) Gene ontology (GO) classification and KEGG pathway analysis of these glycoproteins. (d) Representative MS/MS spectrum of peptide CHEGN # GTFECGACR of β1 with bisecting GlcNAc in MDA/MGAT3 cells. (e) Expression of β1 with bisecting GlcNAc in control and MDA‐231/MGAT3 by immunoprecipitation assay.
    Pha E Agarose, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Lectin histochemical staining profiles in sections of OA cartilage. (a) Binding of PNA to complex chondrons of a severely degenerated cartilage region could be completely blocked with lactose (inset) ascertaining carbohydrate-specific binding. (b) Omission of the incubation step with biotinylated LEA (first-step reagent) from processing excluded probe-independent signal generation. (c, d) ConA staining: staining pattern of MS ≤4 regions included chondrocytes in deep zones of cartilage (c) . Intense staining of matrix and chondrons (inset) in MS ≥9 regions (d) . (e) PSA staining: positivity of chondrons (inset) and matrix, predominantly in MS ≥9 cartilage. (f, g) PHA-E staining: whereas MS ≤4 regions were negative (f) , MS ≥9 areas (g) presented positive chondrons (inset) and matrix. (h) PHA-L staining: binding sites were restricted to chondrons (insert) and matrix of MS ≥9 cartilage. (i-j) VAA staining: whereas the chondrons of MS ≤4 areas were negative (i) , reactivity was observed both in chondrons (inset) and matrix of MS ≥9 cartilage (j) . (k) LEA staining: reactivity for chondrons (inset) and matrix of MS ≥9 regions. (l) MAA-I staining: reactivity included chondrons (inset) and matrix of MS ≥9 cartilage. (m, n) SNA staining: weak staining of matrix and no staining of chondrons in MS ≤4 cartilage (m) , whereas both chondrons (inset) and matrix were positive in MS ≥9 regions (n) . (o) DBA staining: positivity in chondrons (inset) and matrix of MS ≥9 cartilage. (p, q) PNA staining: positive chondrocytes sparely distributed in the deeper zones of MS ≤4 cartilage ( p ; arrows, inset). In MS ≥9 cartilage (q) , intense matrix staining was observed, whereas chondrons were mostly negative (inset). (r) JAC staining: absent in chondrons (inset), but present in superficial zones of MS ≥9 cartilage. Bars in inserts of d,e,g,l,n,p,q,r: 50μm. Bars in inserts of h , j , k , o : 100μm. MS, Mankin score; OA, osteoarthritis.

    Journal: Arthritis Research & Therapy

    Article Title: Glycophenotyping of osteoarthritic cartilage and chondrocytes by RT-qPCR, mass spectrometry, histochemistry with plant/human lectins and lectin localization with a glycoprotein

    doi: 10.1186/ar4330

    Figure Lengend Snippet: Lectin histochemical staining profiles in sections of OA cartilage. (a) Binding of PNA to complex chondrons of a severely degenerated cartilage region could be completely blocked with lactose (inset) ascertaining carbohydrate-specific binding. (b) Omission of the incubation step with biotinylated LEA (first-step reagent) from processing excluded probe-independent signal generation. (c, d) ConA staining: staining pattern of MS ≤4 regions included chondrocytes in deep zones of cartilage (c) . Intense staining of matrix and chondrons (inset) in MS ≥9 regions (d) . (e) PSA staining: positivity of chondrons (inset) and matrix, predominantly in MS ≥9 cartilage. (f, g) PHA-E staining: whereas MS ≤4 regions were negative (f) , MS ≥9 areas (g) presented positive chondrons (inset) and matrix. (h) PHA-L staining: binding sites were restricted to chondrons (insert) and matrix of MS ≥9 cartilage. (i-j) VAA staining: whereas the chondrons of MS ≤4 areas were negative (i) , reactivity was observed both in chondrons (inset) and matrix of MS ≥9 cartilage (j) . (k) LEA staining: reactivity for chondrons (inset) and matrix of MS ≥9 regions. (l) MAA-I staining: reactivity included chondrons (inset) and matrix of MS ≥9 cartilage. (m, n) SNA staining: weak staining of matrix and no staining of chondrons in MS ≤4 cartilage (m) , whereas both chondrons (inset) and matrix were positive in MS ≥9 regions (n) . (o) DBA staining: positivity in chondrons (inset) and matrix of MS ≥9 cartilage. (p, q) PNA staining: positive chondrocytes sparely distributed in the deeper zones of MS ≤4 cartilage ( p ; arrows, inset). In MS ≥9 cartilage (q) , intense matrix staining was observed, whereas chondrons were mostly negative (inset). (r) JAC staining: absent in chondrons (inset), but present in superficial zones of MS ≥9 cartilage. Bars in inserts of d,e,g,l,n,p,q,r: 50μm. Bars in inserts of h , j , k , o : 100μm. MS, Mankin score; OA, osteoarthritis.

    Article Snippet: The plant lectins DBA, JAC, LEA, MAA-I, PHA-E, PHA-L, PNA and SNA were obtained as biotinylated probes from Vector Labs Burlingame, CA, USA.

    Techniques: Staining, Binding Assay, Incubation, Mass Spectrometry

    Electron micrographs of a PHA-L+ type 1 terminal (T1t) making synaptic contact (arrowhead) with the base of a proximal dendrite (den) emerging from an unlabeled putative pyramidal cell perikaryon (Pyr, pk). Scale bar: 0.5 µm.

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: Electron micrographs of a PHA-L+ type 1 terminal (T1t) making synaptic contact (arrowhead) with the base of a proximal dendrite (den) emerging from an unlabeled putative pyramidal cell perikaryon (Pyr, pk). Scale bar: 0.5 µm.

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques:

    PHA-L injections into the basal forebrain mainly label axons in the basolateral amygdalar nucleus. (A) Representative dual injections of PHA-L into the caudal basal forebrain (BF) that involved the ventral pallidum (VP) and substantia innominata (SI)

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: PHA-L injections into the basal forebrain mainly label axons in the basolateral amygdalar nucleus. (A) Representative dual injections of PHA-L into the caudal basal forebrain (BF) that involved the ventral pallidum (VP) and substantia innominata (SI)

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques:

    PHA-L labeled axons in the basolateral nucleus. (A) Photomontage of PHA-L-labeled type 1 axons (black) innervating PV+ interneurons (brown) in the BLa in a field dominated by these axons. Varicosities of type 1 axons are often large and clustered, and

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: PHA-L labeled axons in the basolateral nucleus. (A) Photomontage of PHA-L-labeled type 1 axons (black) innervating PV+ interneurons (brown) in the BLa in a field dominated by these axons. Varicosities of type 1 axons are often large and clustered, and

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques: Labeling

    Triple immunofluorescence labeling for PHA-L, VGAT, and neuronal markers in the BLa. PHA-L+ axons are red, VGAT is green, and neuronal markers (CB in A–E; CaMK in F) are blue. Yellow indicates colocalization of PHA-L and VGAT (i.e. GABAergic axons).

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: Triple immunofluorescence labeling for PHA-L, VGAT, and neuronal markers in the BLa. PHA-L+ axons are red, VGAT is green, and neuronal markers (CB in A–E; CaMK in F) are blue. Yellow indicates colocalization of PHA-L and VGAT (i.e. GABAergic axons).

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques: Immunofluorescence, Labeling

    Synaptic contacts from type 1 PHA-L+ terminals with the perikarya of a PV-immunoreactive interneuron and an unlabeled putative pyramidal neuron (inset in B). (A, B) Schematic drawings, at two levels, of a single parvalbumin-immunoreactive interneuronal

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: Synaptic contacts from type 1 PHA-L+ terminals with the perikarya of a PV-immunoreactive interneuron and an unlabeled putative pyramidal neuron (inset in B). (A, B) Schematic drawings, at two levels, of a single parvalbumin-immunoreactive interneuronal

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques:

    Synaptic contacts from type 1 PHA-L+ terminals onto the proximal dendrites of a PV+ interneuron (inset in B). (A) The proximal end of the lower dendrite of the PV+ interneuron (PVd) receives synaptic input (arrow) from a large PHA-L+ terminal (t1: terminal

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: Synaptic contacts from type 1 PHA-L+ terminals onto the proximal dendrites of a PV+ interneuron (inset in B). (A) The proximal end of the lower dendrite of the PV+ interneuron (PVd) receives synaptic input (arrow) from a large PHA-L+ terminal (t1: terminal

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques:

    (A, B) Electron micrographs of PHA-L+ terminals 2 and 3 (t2, t3) shown in , making synaptic contacts (arrows) onto the PV + interneuron (PVpk) and putative pyramidal neuron (PNpk). Scale bar: 0.5 µm.

    Journal: Neuroscience

    Article Title: POSTSYNAPTIC TARGETS OF GABAERGIC BASAL FOREBRAIN PROJECTIONS TO THE BASOLATERAL AMYGDALA

    doi: 10.1016/j.neuroscience.2011.03.027

    Figure Lengend Snippet: (A, B) Electron micrographs of PHA-L+ terminals 2 and 3 (t2, t3) shown in , making synaptic contacts (arrows) onto the PV + interneuron (PVpk) and putative pyramidal neuron (PNpk). Scale bar: 0.5 µm.

    Article Snippet: Sections through the injection sites and amygdala in PHA-L injected rats were incubated in a rabbit anti-PHA-L antibody (1: 1000; Vector Laboratories Inc.) for 16 h at 4 °C and then processed for the avidin-biotin immunoperoxidase technique using a rabbit Vectastain ABC kit (Vector Laboratories Inc.).

    Techniques:

    (A) Schematic flow of glycoproteomic experiments. Membrane proteins were analyzed by nanoflow-ESI–RPLC–MS/MS before E-PHA capture to verify equivalent protein quality and quantity. Biotinylated E-PHA was added to membrane extracted proteins, and glycoproteins with bisecting glycans were captured using streptavidin magnetic resin prior to nanoflow-ESI–RPLC–MS/MS. Membrane glycoproteins enriched in tumor tissues relative to nonmalignant tissues by E-PHA capture by at least 1.5 times or 150% by spectral counts were identified and are listed in Table 1 . (B) Venn diagram showing the percentage of glycoproteins in Table 1 from endometrioid only (13% purple), serous only (6% blue), or shared (81% blue/purple). (C) Functional annotation of E-PHA enriched membrane glycoproteins using DAVID 2009.

    Journal: Journal of Proteome Research

    Article Title: Glycomic Analysis of Membrane Glycoproteins with Bisecting Glycosylation from Ovarian Cancer Tissues Reveals Novel Structures and Functions

    doi: 10.1021/pr501174p

    Figure Lengend Snippet: (A) Schematic flow of glycoproteomic experiments. Membrane proteins were analyzed by nanoflow-ESI–RPLC–MS/MS before E-PHA capture to verify equivalent protein quality and quantity. Biotinylated E-PHA was added to membrane extracted proteins, and glycoproteins with bisecting glycans were captured using streptavidin magnetic resin prior to nanoflow-ESI–RPLC–MS/MS. Membrane glycoproteins enriched in tumor tissues relative to nonmalignant tissues by E-PHA capture by at least 1.5 times or 150% by spectral counts were identified and are listed in Table 1 . (B) Venn diagram showing the percentage of glycoproteins in Table 1 from endometrioid only (13% purple), serous only (6% blue), or shared (81% blue/purple). (C) Functional annotation of E-PHA enriched membrane glycoproteins using DAVID 2009.

    Article Snippet: Materials Biotinylated E-PHA (Phaseolus vulgaris erythroagglutinin) and streptavidin-HRP were obtained from Vector Laboratories (Burlingame, CA).

    Techniques: Flow Cytometry, Mass Spectrometry, Functional Assay

    Identification of integrin β1 as the target protein bearing bisecting GlcNAc. (a) Intact glycoproteomic analysis by combination of PHA‐E enrichment and LC‐MS/MS (schematic). (b) Venn diagram of numbers of identified glycoproteins bearing bisecting GlcNAc structures in control and MDA‐231/MGAT3. (c) Gene ontology (GO) classification and KEGG pathway analysis of these glycoproteins. (d) Representative MS/MS spectrum of peptide CHEGN # GTFECGACR of β1 with bisecting GlcNAc in MDA/MGAT3 cells. (e) Expression of β1 with bisecting GlcNAc in control and MDA‐231/MGAT3 by immunoprecipitation assay.

    Journal: Journal of Extracellular Vesicles

    Article Title: Bisecting GlcNAc modification diminishes the pro‐metastatic functions of small extracellular vesicles from breast cancer cells, et al. Bisecting GlcNAc modification diminishes the pro‐metastatic functions of small extracellular vesicles from breast cancer cells

    doi: 10.1002/jev2.12005

    Figure Lengend Snippet: Identification of integrin β1 as the target protein bearing bisecting GlcNAc. (a) Intact glycoproteomic analysis by combination of PHA‐E enrichment and LC‐MS/MS (schematic). (b) Venn diagram of numbers of identified glycoproteins bearing bisecting GlcNAc structures in control and MDA‐231/MGAT3. (c) Gene ontology (GO) classification and KEGG pathway analysis of these glycoproteins. (d) Representative MS/MS spectrum of peptide CHEGN # GTFECGACR of β1 with bisecting GlcNAc in MDA/MGAT3 cells. (e) Expression of β1 with bisecting GlcNAc in control and MDA‐231/MGAT3 by immunoprecipitation assay.

    Article Snippet: Eluates were lyophilized, dissolved with binding buffer (50 mM NH4 HCO3 , 150 mM NaCl, 1 mM CaCl2 , 1 mM MnCl2, pH 7.4), and incubated with 50 μl PHA‐E‐agarose (Vector Labs) overnight at 4°C.

    Techniques: Liquid Chromatography with Mass Spectroscopy, Multiple Displacement Amplification, Tandem Mass Spectroscopy, Expressing, Immunoprecipitation

    Expression of bisecting GlcNAc in breast cancer cells. (a) Relative proportions of N‐glycans in human breast cells were calculated by dividing intensity of given type of N‐glycan by total intensity of sample. (b) Bisecting GlcNAc in normal mammary epithelial cells and BC cells evaluated by PHA‐E lectin staining. (c) Bisecting GlcNAc in serum of BC patient evaluated by ELISA. (d) MALDI‐TOF‐MS analysis of N‐glycans from adjacent normal and matched BC tissue. N‐Glycans were divided into two groups (complex and hybrid; high‐mannose type). Relative proportion of bisecting GlcNAc in complex and hybrid is shown. (e) Bisecting GlcNAc in BC tissues in TMA. Immunohistochemistry of representative paired clinical tissues is shown. (f) MGAT3 expression in normal mammary epithelial cells and BC cells evaluated by immunofluorescence. (g) Representative image of MGAT3 expression and bisecting GlcNAc levels in adjacent normal and matched BC tissues. (h) MGAT3 expression in adjacent normal and matched BC tissues, determined by western blotting and Image Pro Plus software program. (i) mRNA expression of MGAT3 in 112 adjacent normal and matched BC tissues in TCGA database. (j) Differential MGAT3 expression of BC tissues in TMA. (k) Overall survival of dichotomized MGAT3 expression in BC patients in TCGA database using GEPIA platform. (l, m) Overall survival of dichotomized MGAT3 expression (l) and bisecting GlcNAc levels (m) in BC patients by TMA

    Journal: Journal of Extracellular Vesicles

    Article Title: Bisecting GlcNAc modification diminishes the pro‐metastatic functions of small extracellular vesicles from breast cancer cells, et al. Bisecting GlcNAc modification diminishes the pro‐metastatic functions of small extracellular vesicles from breast cancer cells

    doi: 10.1002/jev2.12005

    Figure Lengend Snippet: Expression of bisecting GlcNAc in breast cancer cells. (a) Relative proportions of N‐glycans in human breast cells were calculated by dividing intensity of given type of N‐glycan by total intensity of sample. (b) Bisecting GlcNAc in normal mammary epithelial cells and BC cells evaluated by PHA‐E lectin staining. (c) Bisecting GlcNAc in serum of BC patient evaluated by ELISA. (d) MALDI‐TOF‐MS analysis of N‐glycans from adjacent normal and matched BC tissue. N‐Glycans were divided into two groups (complex and hybrid; high‐mannose type). Relative proportion of bisecting GlcNAc in complex and hybrid is shown. (e) Bisecting GlcNAc in BC tissues in TMA. Immunohistochemistry of representative paired clinical tissues is shown. (f) MGAT3 expression in normal mammary epithelial cells and BC cells evaluated by immunofluorescence. (g) Representative image of MGAT3 expression and bisecting GlcNAc levels in adjacent normal and matched BC tissues. (h) MGAT3 expression in adjacent normal and matched BC tissues, determined by western blotting and Image Pro Plus software program. (i) mRNA expression of MGAT3 in 112 adjacent normal and matched BC tissues in TCGA database. (j) Differential MGAT3 expression of BC tissues in TMA. (k) Overall survival of dichotomized MGAT3 expression in BC patients in TCGA database using GEPIA platform. (l, m) Overall survival of dichotomized MGAT3 expression (l) and bisecting GlcNAc levels (m) in BC patients by TMA

    Article Snippet: Eluates were lyophilized, dissolved with binding buffer (50 mM NH4 HCO3 , 150 mM NaCl, 1 mM CaCl2 , 1 mM MnCl2, pH 7.4), and incubated with 50 μl PHA‐E‐agarose (Vector Labs) overnight at 4°C.

    Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Immunofluorescence, Western Blot, Software