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rl 1112  (Vector Laboratories)


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    Vector Laboratories rl 1112
    Rl 1112, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/RL-1112-2/Rhodamine+labeled+Phaseolus+Vulgaris+Leucoagglutinin+(PHA-L)/pm40963256-85-19-17
    Average 93 stars, based on 13 article reviews
    rl 1112 - by Bioz Stars, 2026-10
    93/100 stars

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    Related Articles

    Glycoproteomics:

    Article Title: Targeting GFPT2 to reinvigorate immunotherapy in EGFR-mutated NSCLC
    Article Snippet: Overall N-glycosylation was assessed by incubation with anti-Phaseolus Vulgaris Leucoagglutinin (PHA)-Rhodamine (1:200, RL-1112, Vector) for 1 h at room temperature. .. Specific immune checkpoint ligands and pEGFR glycosylation were probed using primary antibodies against PD-L1 (1:100, ab213524, Abcam), CD276 (1:200, ab134161, Abcam), PVR (1:200, ab103630, Abcam), CD73 (1:200, ab133582, Abcam) and pEGFR (1:500, ab40815, Abcam) overnight at 4 °C, followed by a 1 h incubation with anti-PHA (1:200, RL-1112, Vector). .. Samples were subsequently treated with Cy2-conjugated anti-rabbit secondary antibody (1:200,111-225-144, Jackson ImmunoResearch) for 2 h at room temperature in the dark.

    Incubation:

    Article Title: Targeting GFPT2 to reinvigorate immunotherapy in EGFR-mutated NSCLC
    Article Snippet: Overall N-glycosylation was assessed by incubation with anti-Phaseolus Vulgaris Leucoagglutinin (PHA)-Rhodamine (1:200, RL-1112, Vector) for 1 h at room temperature. .. Specific immune checkpoint ligands and pEGFR glycosylation were probed using primary antibodies against PD-L1 (1:100, ab213524, Abcam), CD276 (1:200, ab134161, Abcam), PVR (1:200, ab103630, Abcam), CD73 (1:200, ab133582, Abcam) and pEGFR (1:500, ab40815, Abcam) overnight at 4 °C, followed by a 1 h incubation with anti-PHA (1:200, RL-1112, Vector). .. Samples were subsequently treated with Cy2-conjugated anti-rabbit secondary antibody (1:200,111-225-144, Jackson ImmunoResearch) for 2 h at room temperature in the dark.

    Article Title: Targeting GFPT2 to reinvigorate immunotherapy in EGFR-mutated NSCLC
    Article Snippet: Sections of 10 μm thickness were prepared using a Leica microtome. .. Overall N-glycosylation was assessed by incubation with anti-Phaseolus Vulgaris Leucoagglutinin (PHA)-Rhodamine (1:200, RL-1112, Vector) for 1 h at room temperature. .. Specific immune checkpoint ligands and pEGFR glycosylation were probed using primary antibodies against PD-L1 (1:100, ab213524, Abcam), CD276 (1:200, ab134161, Abcam), PVR (1:200, ab103630, Abcam), CD73 (1:200, ab133582, Abcam) and pEGFR (1:500, ab40815, Abcam) overnight at 4 °C, followed by a 1 h incubation with anti-PHA (1:200, RL-1112, Vector).

    Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth
    Article Snippet: .. Then, the cells were incubated with PHA-L Rhodamine (1 μg/ml; Vector Laboratories, #RL-1112; RRID: AB_2336657) for 1 hour at 4°C. .. Coverslips were mounted with antifade reagent with DAPI (Invitrogen, # P36935) and visualized with confocal microscopy.

    Article Title: Targeting PGM3 abolishes SREBP-1 activation-hexosamine synthesis feedback regulation to effectively suppress brain tumor growth.
    Article Snippet: .. Then, the cells were incubated with PHA- L Rhodamine (1 μg/ml; Vector Laboratories, #RL- 1112; RRID: AB_2336657) for 1 hour at 4°C. .. Coverslips were mounted with antifade reagent with DAPI (Invitrogen, # P36935) and visualized with confocal microscopy.

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8
    Article Snippet: .. After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos. ..

    other:

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8.
    Article Snippet: Antigen retrieval was done by heat mediation in a citrate buffer (#S1699; Dako), and then sections were blocked with 4% serum for 1 h at room temperature.

    Article Title:
    Article Snippet: Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) , Vector Labs , RL-1112.

    Labeling:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Quadruple Knockout:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Recombinant:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Microscopy:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Software:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Sterility:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Membrane:

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment.
    Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Flag (clone M2, 1:2000) Sigma-Aldrich M1804 Mouse monoclonal anti-GFP (clone 1E10H7, 1:2000) Proteintech 66002-1-Ig Chemicals Gibco Minimum Essential Medium Alpha Invitrogen 12561056 Gibco Penicillin Streptomycin (10,000 U/mL) Invitrogen 15140122 Iron-supplemented bovine calf serum Hyclone SH3007203 Gibco Opti-MEM I Reduced Serum Medium Invitrogen 31985070 Lipofectamine 3000 Invitrogen L3000015 Paraformaldehyde (PFA) Thermo Fisher AC41678-5000 Bovine serum albumin (BSA) Fraction V Dot Scientific DSA30075-100 Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) Vector Labs RL-1112 Nocodazole ThermoFisher AC35824 Cycloheximide AG Scientific C1189 Gibco DMEM, high glucose, HEPES, no phenol red Thermo Fisher 21063029 PNGase F New England Biolabs P0704S Endo H New England Biolabs P0702S 30% acrylamide and bis-acrylamide solution, 37.5:1 Bio-Rad 1610159 N,N,N0,N0-Tetramethylethane-1,2-diamine (TEMED) Thermo Fisher PI17919 Ammonium persulfate (APS) Thermo Fisher BP179-100 Experimental models: Cell lines MAN1 quadruple knockout CHO (QKO MAN1A1/1A2/1B1/1C1) cells Lamriben et al.15 N/A Wild type (WT) parental CHO cells Lamriben et al.15 N/A Recombinant DNA pEGFP-N1-MAN1A1 This paper1 N/A pEGFP-N1-MAN1A1 S12A This paper1 N/A pEGFP-N1-MAN1A1 S12E This paper1 N/A pHEK293Ultra-sHF-LIPA Jin et al.16 N/A (Continued on next page) 4 STAR Protocols 4, 102283, June 16, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Instruments Nikon NIS-Elements C confocal microscope Nikon N/A Software and algorithms Nikon NIS-Elements analysis software Nikon N/A Others Sterile Filter System with PES membrane, 0.45 mm CELLTREAT Scientific Products 229704 HisPurTM Ni-NTA Resin Thermo Fisher 88222 Cytiva Amersham Protran Nitrocellulose Membranes 0.45 mm Thermo Fisher 45-004-002 ll OPEN ACCESSProtocol .. CHO culture medium Reagent Final concentration Amount Gibco Minimum Essential Medium Alpha N/A 500 mL Penicillin-Streptomycin 1% (v/v) 5 mL Note: Store at 4 C for up to 4 weeks.

    Blocking Assay:

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8
    Article Snippet: .. After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos. ..

    Whole Genome Amplification:

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8
    Article Snippet: .. After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos. ..



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    The ileal epithelium is dominated by L-PHA + , complex N-glycans in healthy subjects and by truncated, sWGA + N-glycans in active Crohn disease (A) Schematic of N-glycan branching cascade demonstrating the glycan targets of lectins and manganese (Mn) utilization. sWGA (green) stains terminal GlcNAc residues. L-PHA (red) stains complex tri- and tetra-antennary N-glycans. Relative UDP-GlcNAc affinity of glycosyltransferases is also represented (300-fold change from Mgat1 to Mgat5). Figure generated in Biorender, informed by Cummings et al. (ref. ). (B) Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissue paraffin sections, stained for PHA-L (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with <t>fluorescein</t> dyes (PHA-L 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. n = 3–4. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Ratio of sWGA/L-PHA also depicted. Individual data points, mean, and SEM graphed with statistical significance are determined by t test, with p value indicated by two asterisks (<0.01) or three asterisks (<0.001). (C) Volcano plot of glycosyltransferase genes differentially expressed in ileal mucosal biopsies from male and female pediatric patients with severe ileal Crohn disease compared to non-IBD controls. This is a secondary analysis of RNA-seq data (GEO: GSE57945 ). n = 42 controls, n = 63 CD patients. There is an enrichment for MGAT-related pathway members with increased expression of MGAT1 and decreased expression of MGAT3 , MGAT4A/4B , and MGAT5 . MGAT4B is the most highly expressed gene .
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    The ileal epithelium is dominated by L-PHA + , complex N-glycans in healthy subjects and by truncated, sWGA + N-glycans in active Crohn disease (A) Schematic of N-glycan branching cascade demonstrating the glycan targets of lectins and manganese (Mn) utilization. sWGA (green) stains terminal GlcNAc residues. L-PHA (red) stains complex tri- and tetra-antennary N-glycans. Relative UDP-GlcNAc affinity of glycosyltransferases is also represented (300-fold change from Mgat1 to Mgat5). Figure generated in Biorender, informed by Cummings et al. (ref. ). (B) Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissue paraffin sections, stained for PHA-L (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with <t>fluorescein</t> dyes (PHA-L 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. n = 3–4. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Ratio of sWGA/L-PHA also depicted. Individual data points, mean, and SEM graphed with statistical significance are determined by t test, with p value indicated by two asterisks (<0.01) or three asterisks (<0.001). (C) Volcano plot of glycosyltransferase genes differentially expressed in ileal mucosal biopsies from male and female pediatric patients with severe ileal Crohn disease compared to non-IBD controls. This is a secondary analysis of RNA-seq data (GEO: GSE57945 ). n = 42 controls, n = 63 CD patients. There is an enrichment for MGAT-related pathway members with increased expression of MGAT1 and decreased expression of MGAT3 , MGAT4A/4B , and MGAT5 . MGAT4B is the most highly expressed gene .
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    Image Search Results


    The ileal epithelium is dominated by L-PHA + , complex N-glycans in healthy subjects and by truncated, sWGA + N-glycans in active Crohn disease (A) Schematic of N-glycan branching cascade demonstrating the glycan targets of lectins and manganese (Mn) utilization. sWGA (green) stains terminal GlcNAc residues. L-PHA (red) stains complex tri- and tetra-antennary N-glycans. Relative UDP-GlcNAc affinity of glycosyltransferases is also represented (300-fold change from Mgat1 to Mgat5). Figure generated in Biorender, informed by Cummings et al. (ref. ). (B) Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissue paraffin sections, stained for PHA-L (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (PHA-L 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. n = 3–4. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Ratio of sWGA/L-PHA also depicted. Individual data points, mean, and SEM graphed with statistical significance are determined by t test, with p value indicated by two asterisks (<0.01) or three asterisks (<0.001). (C) Volcano plot of glycosyltransferase genes differentially expressed in ileal mucosal biopsies from male and female pediatric patients with severe ileal Crohn disease compared to non-IBD controls. This is a secondary analysis of RNA-seq data (GEO: GSE57945 ). n = 42 controls, n = 63 CD patients. There is an enrichment for MGAT-related pathway members with increased expression of MGAT1 and decreased expression of MGAT3 , MGAT4A/4B , and MGAT5 . MGAT4B is the most highly expressed gene .

    Journal: Human Genetics and Genomics Advances

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

    doi: 10.1016/j.xhgg.2025.100517

    Figure Lengend Snippet: The ileal epithelium is dominated by L-PHA + , complex N-glycans in healthy subjects and by truncated, sWGA + N-glycans in active Crohn disease (A) Schematic of N-glycan branching cascade demonstrating the glycan targets of lectins and manganese (Mn) utilization. sWGA (green) stains terminal GlcNAc residues. L-PHA (red) stains complex tri- and tetra-antennary N-glycans. Relative UDP-GlcNAc affinity of glycosyltransferases is also represented (300-fold change from Mgat1 to Mgat5). Figure generated in Biorender, informed by Cummings et al. (ref. ). (B) Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissue paraffin sections, stained for PHA-L (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (PHA-L 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. n = 3–4. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Ratio of sWGA/L-PHA also depicted. Individual data points, mean, and SEM graphed with statistical significance are determined by t test, with p value indicated by two asterisks (<0.01) or three asterisks (<0.001). (C) Volcano plot of glycosyltransferase genes differentially expressed in ileal mucosal biopsies from male and female pediatric patients with severe ileal Crohn disease compared to non-IBD controls. This is a secondary analysis of RNA-seq data (GEO: GSE57945 ). n = 42 controls, n = 63 CD patients. There is an enrichment for MGAT-related pathway members with increased expression of MGAT1 and decreased expression of MGAT3 , MGAT4A/4B , and MGAT5 . MGAT4B is the most highly expressed gene .

    Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

    Techniques: Glycoproteomics, Generated, Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence, RNA Sequencing, Expressing

    Truncated sWGA + N-glycans are increased at the apical/brush border of ileal epithelial cells in ZIP8 391-Thr carriers with Crohn disease Representative lectin immunofluorescence images of ileal biopsies of genotyped patients with active Crohn’s ileitis in (A) ZIP8 391A/391A (non-carriers) and (B) ZIP8 391A/391T (ZIP8 391-Thr heterozygous carriers). Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Scale bar: 50 μm. Asterisk highlights enhanced localization of sWGA staining to the apical membrane/glycocalyx of epithelial cells. To focus on the epithelial compartment, sWGA and L-PHA distribution and overlap were blindly scored by two investigators. Representative images from n = 3 patients/genotype included in figure; histology reviewed and scored for n = 9 ZIP8 391A/391A and n = 6 ZIP8 391A/391T individuals. ns, not statistically significant.

    Journal: Human Genetics and Genomics Advances

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

    doi: 10.1016/j.xhgg.2025.100517

    Figure Lengend Snippet: Truncated sWGA + N-glycans are increased at the apical/brush border of ileal epithelial cells in ZIP8 391-Thr carriers with Crohn disease Representative lectin immunofluorescence images of ileal biopsies of genotyped patients with active Crohn’s ileitis in (A) ZIP8 391A/391A (non-carriers) and (B) ZIP8 391A/391T (ZIP8 391-Thr heterozygous carriers). Confocal laser-scanning triple-label immunofluorescence microscopy images of human ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Fluorescence intensity of sWGA and L-PHA normalized to Hoechst was measured using Metamorph to compare healthy individuals and those with Crohn disease (CD). Scale bar: 50 μm. Asterisk highlights enhanced localization of sWGA staining to the apical membrane/glycocalyx of epithelial cells. To focus on the epithelial compartment, sWGA and L-PHA distribution and overlap were blindly scored by two investigators. Representative images from n = 3 patients/genotype included in figure; histology reviewed and scored for n = 9 ZIP8 391A/391A and n = 6 ZIP8 391A/391T individuals. ns, not statistically significant.

    Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

    Techniques: Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence, Membrane

    ZIP8 391-Thr-associated defect in N-glycosylation in the ileal epithelial compartment is recapitulated in Zip8 393T-knockin mice (A) Confocal laser-scanning triple-label immunofluorescence microscopy images of Zip8 +/+ (WT), Zip8 +/393T (Het), and Zip8 393T/393T (HM) ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. N = 5 male and female mice/genotype with n = 3–5 fields of view/mice imaged. (B) Quantification of sWGA and L-PHA fluorescence intensity normalized to Hoechst measured using Metamorph. Statistical significance determined by one-way ANOVA, p value indicated by one asterisk (<0.05) or three asterisks (<0.001). (C) Averaged spectra of matrix-associated laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) following on-tissue PNGase F digest to measure the differential abundance of N-glycan species in transverse section of distal ileal tissue of Zip8 +/+ and Zip8 393T/393T mice. Higher m/z species represent tri- and tetra-antennary N-glycan branching. N = 4 male mice/genotype. (D) Confocal laser-scanning triple-label immunofluorescence microscopy images of Jackson C57BL/6 male mice fed purified diet containing variable Mn (<1 ppm = Mn deficient and 2,400 ppm = Mn excess) for 4 weeks. Ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. N = 3 male mice in each group, 2–3 fields of view imaged and quantified per mouse. Scale bar: 50 μm. Individual data points, mean, and SEM graphed, with statistical significance determined by one-way ANOVA and p value indicated by two asterisks (<0.01) or three asterisks (<0.001).

    Journal: Human Genetics and Genomics Advances

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

    doi: 10.1016/j.xhgg.2025.100517

    Figure Lengend Snippet: ZIP8 391-Thr-associated defect in N-glycosylation in the ileal epithelial compartment is recapitulated in Zip8 393T-knockin mice (A) Confocal laser-scanning triple-label immunofluorescence microscopy images of Zip8 +/+ (WT), Zip8 +/393T (Het), and Zip8 393T/393T (HM) ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. Scale bar: 50 μm. N = 5 male and female mice/genotype with n = 3–5 fields of view/mice imaged. (B) Quantification of sWGA and L-PHA fluorescence intensity normalized to Hoechst measured using Metamorph. Statistical significance determined by one-way ANOVA, p value indicated by one asterisk (<0.05) or three asterisks (<0.001). (C) Averaged spectra of matrix-associated laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) following on-tissue PNGase F digest to measure the differential abundance of N-glycan species in transverse section of distal ileal tissue of Zip8 +/+ and Zip8 393T/393T mice. Higher m/z species represent tri- and tetra-antennary N-glycan branching. N = 4 male mice/genotype. (D) Confocal laser-scanning triple-label immunofluorescence microscopy images of Jackson C57BL/6 male mice fed purified diet containing variable Mn (<1 ppm = Mn deficient and 2,400 ppm = Mn excess) for 4 weeks. Ileal tissues paraffin sections, stained for L-PHA (red), sWGA (green), Hoechst (blue), and merged image. N = 3 male mice in each group, 2–3 fields of view imaged and quantified per mouse. Scale bar: 50 μm. Individual data points, mean, and SEM graphed, with statistical significance determined by one-way ANOVA and p value indicated by two asterisks (<0.01) or three asterisks (<0.001).

    Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

    Techniques: Glycoproteomics, Knock-In, Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence, Mass Spectrometry, Imaging, Purification

    Oral GlcNAc supplementation restores complex N-glycan branching in intestinal epithelial cells in Zip8 +/393T and Zip8 393T/393T mice Confocal laser-scanning triple-label immunofluorescence microscopy images of Zip8 +/+ (WT) (A), Zip8 +/393T (Het) (B), and Zip8 393T/393T (HM) (C) mice ileal tissue paraffin sections, stained for sWGA (green), L-PHA (red), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. L-PHA and sWGA fluorescence intensity measured using Metamorph. Scale bar: 50 μm. N = 4–5 male and female mice/genotype, with n = 3–7 fields of view/mice imaged. Individual data points, mean, and SEM are graphed. Statistical significance was determined by Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons testing; p value indicated by one asterisk (<0.05), two asterisks (<0.01), three asterisks (<0.001), or four asterisks (<0.0001).

    Journal: Human Genetics and Genomics Advances

    Article Title: Aberrant N-glycosylation may be a therapeutic target in carriers of a common and highly pleiotropic variant in the manganese transporter ZIP8

    doi: 10.1016/j.xhgg.2025.100517

    Figure Lengend Snippet: Oral GlcNAc supplementation restores complex N-glycan branching in intestinal epithelial cells in Zip8 +/393T and Zip8 393T/393T mice Confocal laser-scanning triple-label immunofluorescence microscopy images of Zip8 +/+ (WT) (A), Zip8 +/393T (Het) (B), and Zip8 393T/393T (HM) (C) mice ileal tissue paraffin sections, stained for sWGA (green), L-PHA (red), Hoechst (blue), and merged image. Samples were incubated with fluorescein dyes (L-PHA 639, sWGA 488, and Hoechst 405), 10 μg/mL, in blocking buffer for 1 h at room temperature. L-PHA and sWGA fluorescence intensity measured using Metamorph. Scale bar: 50 μm. N = 4–5 male and female mice/genotype, with n = 3–7 fields of view/mice imaged. Individual data points, mean, and SEM are graphed. Statistical significance was determined by Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons testing; p value indicated by one asterisk (<0.05), two asterisks (<0.01), three asterisks (<0.001), or four asterisks (<0.0001).

    Article Snippet: After blocking, samples were incubated with fluorescein dyes ( Phaseolus vulgaris leucoagglutinin [PHA-L] rhodamine-conjugated lectin and succinylated wheat germ agglutinin [WGA] lectin) from Vector Labs (catalog nos.

    Techniques: Glycoproteomics, Immunofluorescence, Microscopy, Staining, Incubation, Blocking Assay, Fluorescence

    Journal: STAR Protocols

    Article Title: Analysis of mannosidase I activity in interphase and mitotic cells by lectin staining and endoglycosidase H treatment

    doi: 10.1016/j.xpro.2023.102283

    Figure Lengend Snippet:

    Article Snippet: Rhodamine labeled phaseolus vulgaris leucoagglutinin (PHA-L) , Vector Labs , RL-1112.

    Techniques: Labeling, Plasmid Preparation, Quadruple Knockout, Recombinant, Microscopy, Software, Sterility, Membrane