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concentrated mncl 2 solution  (New England Biolabs)


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    New England Biolabs concentrated mncl 2 solution
    Concentrated Mncl 2 Solution, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2943 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/lambda+protein+phosphatase+buffer/Lambda+Protein+Phosphatase/pmc03538215-190-21-25
    Average 99 stars, based on 2943 article reviews
    concentrated mncl 2 solution - by Bioz Stars, 2026-11
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    Related Articles

    Incubation:

    Article Title: AKT Phosphorylation of FOXK2 at Ser398 Impacts Breast Cancer Cell Growth and Viability
    Article Snippet: .. Afterwards, 100 μg of protein were incubated in a total of 50 μL containing 400 units of lambda protein phosphatase (λPP; P0753S, New England BioLabs Inc., Ipswich, MA, USA), 1× PMP (NEBuffer Pack for Protein MetalloPhosphatases; B0761S, New England BioLabs Inc.) and 1 mM MnCl2 (B1761S, New England BioLabs Inc.) for 1 h at 37°C. ..

    Article Title: Single-molecule resolution of post-translational modification on GRK4 with cyclodextrin/SiN x hybrid nanopore.
    Article Snippet: .. A 100 μL of the mixture containing 10 μM of pep-T182-P and 200 U of lambda protein phosphatase in 1 mM MnCl2 and NEbuffer for PMP was incubated at 30 ◦C for 2 h, a control assay is under identical condition but without lambda protein phosphatase. ..

    Article Title: Identification of NEK9 as a novel substrate of protein kinase B.
    Article Snippet: Protein kinase B (PKB/Akt) regulates diverse cellular processes through phosphorylation of numerous substrate proteins downstream of phosphatidylinositol 3-kinase (PI3K).. Although many PKB substrates have been identified, additional substrates remain to be characterized.. NEK9 is an essential serine/threonine kinase involved in mitotic progression and contains two putative PKB consensus phosphorylation motifs.

    Article Title: The PvAURK-PvGRFs regulatory module controls salt tolerance in common bean (Phaseolus vulgaris L.).
    Article Snippet: .. For the dephosphorylation assay, the extracted protein samples were incubated with λ-protein phosphatase (λ-PPase, New England Biolabs) at 30 ◦C for 45 min according to the manufacturer's instructions. ..

    Article Title: VASH1 phosphorylation regulates tubulin detyrosination via CNP/PRKG1α signaling in DRG neurons
    Article Snippet: .. For phosphatase treatment experiments, bead-bound complexes were incubated with phosphatase (Lambda protein phosphatase, New England Biolabs) for 30 min at 30°C according to the manufacturer’s protocol before elution. ..

    Control:

    Article Title: Single-molecule resolution of post-translational modification on GRK4 with cyclodextrin/SiN x hybrid nanopore.
    Article Snippet: .. A 100 μL of the mixture containing 10 μM of pep-T182-P and 200 U of lambda protein phosphatase in 1 mM MnCl2 and NEbuffer for PMP was incubated at 30 ◦C for 2 h, a control assay is under identical condition but without lambda protein phosphatase. ..

    Thin Layer Chromatography:

    Article Title: A bifunctional ARHGEF17-AS1 locus encodes MSEP, a protein that supports mitotic spindle organization.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-α-tubulin (clone DM1A) Sigma-Aldrich Cat# T6199; RRID: AB_477583 Rabbit polyclonal anti-α-tubulin Proteintech Cat# 11224-1-AP; RRID: AB_2210206 Mouse monoclonal anti-γ-tubulin (clone GTU-88) Sigma-Aldrich Cat# T5326; RRID: AB_532292 Rabbit polyclonal anti-γ-tubulin Sigma-Aldrich Cat# T5192; RRID: AB_261690 Mouse monoclonal anti-RPS3 (clone 2G7H4) Proteintech Cat# 66046-1-Ig; RRID: AB_11182493 Rabbit polyclonal anti-RPS3 Novus Biologicals Cat# NBP1-33691; RRID: AB_2180759 Rabbit monoclonal anti-RPS3 (clone D50G7) Cell Signaling Technology Cat# 9538S; RRID: AB_10622028 Rabbit polyclonal anti-RNF10 Proteintech Cat# 16936-1-AP; RRID: AB_2181846 Mouse monoclonal anti-TPX2 (clone 2E10C1) Proteintech Cat# 68712-1-Ig; RRID: AB_3670405 Rabbit polyclonal anti-TPX2 Proteintech Cat# 11741-1-AP; RRID: AB_2208895 Mouse monoclonal anti-KPNA2 (clone 2G9C1) Proteintech Cat# 66870-1-Ig; RRID: AB_2882207 Rabbit polyclonal anti-KPNA2 Proteintech Cat# 10819-1-AP; RRID: AB_2265526 Rabbit polyclonal anti-DYNC1H1/dynein heavy chain Proteintech Cat# 12345-1-AP; RRID: AB_2261765 Mouse monoclonal anti-cytoplasmic dynein intermediate chain (clone 74.1) Abcam Cat# ab23905; RRID: AB_2096669 Mouse monoclonal anti-FLAG M2 (clone M2) Sigma-Aldrich Cat# F1804; RRID: AB_262044 Mouse monoclonal anti-GFP tag (clone 1E10H7) Proteintech Cat# 66002-1-Ig; RRID: AB_11182611 Rabbit polyclonal anti-GFP Abcam Cat# ab290; RRID: AB_303395 Rabbit monoclonal anti-GAPDH (clone D16H11) Cell Signaling Technology Cat# 5174; RRID: AB_10622025 Rabbit monoclonal anti-Aurora-A/AIK (clone 1G4) Cell Signaling Technology Cat# 4718; RRID: AB_2061482 Mouse monoclonal anti-Aurora-A (clone 1F4B10) Proteintech Cat# 66757-1-Ig; RRID: AB_2882103 Rabbit monoclonal anti-WEE1 (clone D10D2) Cell Signaling Technology Cat# 13084; RRID: AB_2713924 Rabbit monoclonal anti-PLK1 (clone 208G4) Cell Signaling Technology Cat# 4513; RRID: AB_2167409 Rabbit polyclonal anti-vinculin Proteintech Cat# 26520-1-AP; RRID: AB_2868558 Normal mouse IgG MilliporeSigma Cat# 12–371; RRID: AB_145840 Normal rabbit IgG MilliporeSigma Cat# 12–370; RRID: AB_145841 Rabbit polyclonal anti-MSEP; immunogen CGHRPQRLHPGRP (residues 257–269) This paper; Innovagen AB N/A Anti-MYC tag antibody Abcam Cat# ab32; RRID: AB_303599 Anti-cyclin B1 antibody Abcam Cat# ab32053; RRID: AB_731779 Anti-total PARP antibody Abcam Cat# ab191217; RRID: AB_2861274 (Continued on next page) 16 Cell Reports 45, 118026, October 27, 2026 .. REAGENT or RESOURCE SOURCE IDENTIFIER N-Acetyl-D-sphingosine; C2 ceramide (d18:1/2:0), ≥97% (TLC) Sigma-Aldrich Cat# A7191; CAS: 3102–57-6 Lambda Protein Phosphatase New England Biolabs Cat# P0753S Puromycin dihydrochloride Sigma-Aldrich Cat# P8833; CAS: 58–58-2 Hygromycin B Sigma-Aldrich Cat# H3274; CAS: 31282–04-9 TRIzol Reagent Invitrogen/Thermo Fisher Scientific Cat# 15596026 ProLong Gold Antifade Mountant Invitrogen/Thermo Fisher Scientific Cat# P36930 RNase A Invitrogen/Thermo Fisher Scientific Cat# 12091021 Halt Protease and Phosphatase Inhibitor Cocktail Thermo Fisher Scientific Cat# 78440 Phosphatase Inhibitor Cocktail Set IV MilliporeSigma Cat# 524628 Protein G magnetic beads Invitrogen/Thermo Fisher Scientific Cat# 10004D MSEP immunogen peptide CGHRPQRLHPGRP (residues 257–269) This paper; Innovagen AB N/A Critical commercial assays Lipofectamine RNAiMAX Transfection Reagent Invitrogen/Thermo Fisher Scientific Cat# 13778150 Lipofectamine 2000 Transfection Reagent Invitrogen/Thermo Fisher Scientific Cat# 11668019 SF Cell Line 4D-Nucleofector X Kit L Lonza Cat# V4XC-2012 ReliaPrep RNA Cell Miniprep System Promega Cat# Z6010, Z6011. .. ImProm-II Reverse Transcription System Promega Cat# A3800 Power SYBR Green PCR Master Mix Applied Biosystems/Thermo Fisher Scientific Cat# 4367659 RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics/Bio-Techne Cat# 323100 Duolink In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23225 NuPAGE Novex 4–12% Bis-Tris protein gels Invitrogen/Thermo Fisher Scientific Cat# NP0321 Microcon 30 kDa centrifugal filter units Merck Millipore Cat# MRCF0R030 CellTiter-Glo 2.0 Cell Viability Assay Promega Cat# G9241 Deposited data RNA-sequencing data reported in this study This paper; NCBI Gene Expression Omnibus GEO: GSE274156; https://www.ncbi.nlm.

    Magnetic Beads:

    Article Title: A bifunctional ARHGEF17-AS1 locus encodes MSEP, a protein that supports mitotic spindle organization.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-α-tubulin (clone DM1A) Sigma-Aldrich Cat# T6199; RRID: AB_477583 Rabbit polyclonal anti-α-tubulin Proteintech Cat# 11224-1-AP; RRID: AB_2210206 Mouse monoclonal anti-γ-tubulin (clone GTU-88) Sigma-Aldrich Cat# T5326; RRID: AB_532292 Rabbit polyclonal anti-γ-tubulin Sigma-Aldrich Cat# T5192; RRID: AB_261690 Mouse monoclonal anti-RPS3 (clone 2G7H4) Proteintech Cat# 66046-1-Ig; RRID: AB_11182493 Rabbit polyclonal anti-RPS3 Novus Biologicals Cat# NBP1-33691; RRID: AB_2180759 Rabbit monoclonal anti-RPS3 (clone D50G7) Cell Signaling Technology Cat# 9538S; RRID: AB_10622028 Rabbit polyclonal anti-RNF10 Proteintech Cat# 16936-1-AP; RRID: AB_2181846 Mouse monoclonal anti-TPX2 (clone 2E10C1) Proteintech Cat# 68712-1-Ig; RRID: AB_3670405 Rabbit polyclonal anti-TPX2 Proteintech Cat# 11741-1-AP; RRID: AB_2208895 Mouse monoclonal anti-KPNA2 (clone 2G9C1) Proteintech Cat# 66870-1-Ig; RRID: AB_2882207 Rabbit polyclonal anti-KPNA2 Proteintech Cat# 10819-1-AP; RRID: AB_2265526 Rabbit polyclonal anti-DYNC1H1/dynein heavy chain Proteintech Cat# 12345-1-AP; RRID: AB_2261765 Mouse monoclonal anti-cytoplasmic dynein intermediate chain (clone 74.1) Abcam Cat# ab23905; RRID: AB_2096669 Mouse monoclonal anti-FLAG M2 (clone M2) Sigma-Aldrich Cat# F1804; RRID: AB_262044 Mouse monoclonal anti-GFP tag (clone 1E10H7) Proteintech Cat# 66002-1-Ig; RRID: AB_11182611 Rabbit polyclonal anti-GFP Abcam Cat# ab290; RRID: AB_303395 Rabbit monoclonal anti-GAPDH (clone D16H11) Cell Signaling Technology Cat# 5174; RRID: AB_10622025 Rabbit monoclonal anti-Aurora-A/AIK (clone 1G4) Cell Signaling Technology Cat# 4718; RRID: AB_2061482 Mouse monoclonal anti-Aurora-A (clone 1F4B10) Proteintech Cat# 66757-1-Ig; RRID: AB_2882103 Rabbit monoclonal anti-WEE1 (clone D10D2) Cell Signaling Technology Cat# 13084; RRID: AB_2713924 Rabbit monoclonal anti-PLK1 (clone 208G4) Cell Signaling Technology Cat# 4513; RRID: AB_2167409 Rabbit polyclonal anti-vinculin Proteintech Cat# 26520-1-AP; RRID: AB_2868558 Normal mouse IgG MilliporeSigma Cat# 12–371; RRID: AB_145840 Normal rabbit IgG MilliporeSigma Cat# 12–370; RRID: AB_145841 Rabbit polyclonal anti-MSEP; immunogen CGHRPQRLHPGRP (residues 257–269) This paper; Innovagen AB N/A Anti-MYC tag antibody Abcam Cat# ab32; RRID: AB_303599 Anti-cyclin B1 antibody Abcam Cat# ab32053; RRID: AB_731779 Anti-total PARP antibody Abcam Cat# ab191217; RRID: AB_2861274 (Continued on next page) 16 Cell Reports 45, 118026, October 27, 2026 .. REAGENT or RESOURCE SOURCE IDENTIFIER N-Acetyl-D-sphingosine; C2 ceramide (d18:1/2:0), ≥97% (TLC) Sigma-Aldrich Cat# A7191; CAS: 3102–57-6 Lambda Protein Phosphatase New England Biolabs Cat# P0753S Puromycin dihydrochloride Sigma-Aldrich Cat# P8833; CAS: 58–58-2 Hygromycin B Sigma-Aldrich Cat# H3274; CAS: 31282–04-9 TRIzol Reagent Invitrogen/Thermo Fisher Scientific Cat# 15596026 ProLong Gold Antifade Mountant Invitrogen/Thermo Fisher Scientific Cat# P36930 RNase A Invitrogen/Thermo Fisher Scientific Cat# 12091021 Halt Protease and Phosphatase Inhibitor Cocktail Thermo Fisher Scientific Cat# 78440 Phosphatase Inhibitor Cocktail Set IV MilliporeSigma Cat# 524628 Protein G magnetic beads Invitrogen/Thermo Fisher Scientific Cat# 10004D MSEP immunogen peptide CGHRPQRLHPGRP (residues 257–269) This paper; Innovagen AB N/A Critical commercial assays Lipofectamine RNAiMAX Transfection Reagent Invitrogen/Thermo Fisher Scientific Cat# 13778150 Lipofectamine 2000 Transfection Reagent Invitrogen/Thermo Fisher Scientific Cat# 11668019 SF Cell Line 4D-Nucleofector X Kit L Lonza Cat# V4XC-2012 ReliaPrep RNA Cell Miniprep System Promega Cat# Z6010, Z6011. .. ImProm-II Reverse Transcription System Promega Cat# A3800 Power SYBR Green PCR Master Mix Applied Biosystems/Thermo Fisher Scientific Cat# 4367659 RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics/Bio-Techne Cat# 323100 Duolink In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23225 NuPAGE Novex 4–12% Bis-Tris protein gels Invitrogen/Thermo Fisher Scientific Cat# NP0321 Microcon 30 kDa centrifugal filter units Merck Millipore Cat# MRCF0R030 CellTiter-Glo 2.0 Cell Viability Assay Promega Cat# G9241 Deposited data RNA-sequencing data reported in this study This paper; NCBI Gene Expression Omnibus GEO: GSE274156; https://www.ncbi.nlm.

    Transfection:

    Article Title: A bifunctional ARHGEF17-AS1 locus encodes MSEP, a protein that supports mitotic spindle organization.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-α-tubulin (clone DM1A) Sigma-Aldrich Cat# T6199; RRID: AB_477583 Rabbit polyclonal anti-α-tubulin Proteintech Cat# 11224-1-AP; RRID: AB_2210206 Mouse monoclonal anti-γ-tubulin (clone GTU-88) Sigma-Aldrich Cat# T5326; RRID: AB_532292 Rabbit polyclonal anti-γ-tubulin Sigma-Aldrich Cat# T5192; RRID: AB_261690 Mouse monoclonal anti-RPS3 (clone 2G7H4) Proteintech Cat# 66046-1-Ig; RRID: AB_11182493 Rabbit polyclonal anti-RPS3 Novus Biologicals Cat# NBP1-33691; RRID: AB_2180759 Rabbit monoclonal anti-RPS3 (clone D50G7) Cell Signaling Technology Cat# 9538S; RRID: AB_10622028 Rabbit polyclonal anti-RNF10 Proteintech Cat# 16936-1-AP; RRID: AB_2181846 Mouse monoclonal anti-TPX2 (clone 2E10C1) Proteintech Cat# 68712-1-Ig; RRID: AB_3670405 Rabbit polyclonal anti-TPX2 Proteintech Cat# 11741-1-AP; RRID: AB_2208895 Mouse monoclonal anti-KPNA2 (clone 2G9C1) Proteintech Cat# 66870-1-Ig; RRID: AB_2882207 Rabbit polyclonal anti-KPNA2 Proteintech Cat# 10819-1-AP; RRID: AB_2265526 Rabbit polyclonal anti-DYNC1H1/dynein heavy chain Proteintech Cat# 12345-1-AP; RRID: AB_2261765 Mouse monoclonal anti-cytoplasmic dynein intermediate chain (clone 74.1) Abcam Cat# ab23905; RRID: AB_2096669 Mouse monoclonal anti-FLAG M2 (clone M2) Sigma-Aldrich Cat# F1804; RRID: AB_262044 Mouse monoclonal anti-GFP tag (clone 1E10H7) Proteintech Cat# 66002-1-Ig; RRID: AB_11182611 Rabbit polyclonal anti-GFP Abcam Cat# ab290; RRID: AB_303395 Rabbit monoclonal anti-GAPDH (clone D16H11) Cell Signaling Technology Cat# 5174; RRID: AB_10622025 Rabbit monoclonal anti-Aurora-A/AIK (clone 1G4) Cell Signaling Technology Cat# 4718; RRID: AB_2061482 Mouse monoclonal anti-Aurora-A (clone 1F4B10) Proteintech Cat# 66757-1-Ig; RRID: AB_2882103 Rabbit monoclonal anti-WEE1 (clone D10D2) Cell Signaling Technology Cat# 13084; RRID: AB_2713924 Rabbit monoclonal anti-PLK1 (clone 208G4) Cell Signaling Technology Cat# 4513; RRID: AB_2167409 Rabbit polyclonal anti-vinculin Proteintech Cat# 26520-1-AP; RRID: AB_2868558 Normal mouse IgG MilliporeSigma Cat# 12–371; RRID: AB_145840 Normal rabbit IgG MilliporeSigma Cat# 12–370; RRID: AB_145841 Rabbit polyclonal anti-MSEP; immunogen CGHRPQRLHPGRP (residues 257–269) This paper; Innovagen AB N/A Anti-MYC tag antibody Abcam Cat# ab32; RRID: AB_303599 Anti-cyclin B1 antibody Abcam Cat# ab32053; RRID: AB_731779 Anti-total PARP antibody Abcam Cat# ab191217; RRID: AB_2861274 (Continued on next page) 16 Cell Reports 45, 118026, October 27, 2026 .. REAGENT or RESOURCE SOURCE IDENTIFIER N-Acetyl-D-sphingosine; C2 ceramide (d18:1/2:0), ≥97% (TLC) Sigma-Aldrich Cat# A7191; CAS: 3102–57-6 Lambda Protein Phosphatase New England Biolabs Cat# P0753S Puromycin dihydrochloride Sigma-Aldrich Cat# P8833; CAS: 58–58-2 Hygromycin B Sigma-Aldrich Cat# H3274; CAS: 31282–04-9 TRIzol Reagent Invitrogen/Thermo Fisher Scientific Cat# 15596026 ProLong Gold Antifade Mountant Invitrogen/Thermo Fisher Scientific Cat# P36930 RNase A Invitrogen/Thermo Fisher Scientific Cat# 12091021 Halt Protease and Phosphatase Inhibitor Cocktail Thermo Fisher Scientific Cat# 78440 Phosphatase Inhibitor Cocktail Set IV MilliporeSigma Cat# 524628 Protein G magnetic beads Invitrogen/Thermo Fisher Scientific Cat# 10004D MSEP immunogen peptide CGHRPQRLHPGRP (residues 257–269) This paper; Innovagen AB N/A Critical commercial assays Lipofectamine RNAiMAX Transfection Reagent Invitrogen/Thermo Fisher Scientific Cat# 13778150 Lipofectamine 2000 Transfection Reagent Invitrogen/Thermo Fisher Scientific Cat# 11668019 SF Cell Line 4D-Nucleofector X Kit L Lonza Cat# V4XC-2012 ReliaPrep RNA Cell Miniprep System Promega Cat# Z6010, Z6011. .. ImProm-II Reverse Transcription System Promega Cat# A3800 Power SYBR Green PCR Master Mix Applied Biosystems/Thermo Fisher Scientific Cat# 4367659 RNAscope Multiplex Fluorescent Reagent Kit v2 Advanced Cell Diagnostics/Bio-Techne Cat# 323100 Duolink In Situ Red Starter Kit Mouse/Rabbit MilliporeSigma Cat# DUO92101 Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23225 NuPAGE Novex 4–12% Bis-Tris protein gels Invitrogen/Thermo Fisher Scientific Cat# NP0321 Microcon 30 kDa centrifugal filter units Merck Millipore Cat# MRCF0R030 CellTiter-Glo 2.0 Cell Viability Assay Promega Cat# G9241 Deposited data RNA-sequencing data reported in this study This paper; NCBI Gene Expression Omnibus GEO: GSE274156; https://www.ncbi.nlm.

    De-Phosphorylation Assay:

    Article Title: The PvAURK-PvGRFs regulatory module controls salt tolerance in common bean (Phaseolus vulgaris L.).
    Article Snippet: .. For the dephosphorylation assay, the extracted protein samples were incubated with λ-protein phosphatase (λ-PPase, New England Biolabs) at 30 ◦C for 45 min according to the manufacturer's instructions. ..



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    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with <t>λ-phosphatase</t> for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .
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    lambda phosphatase assay buffer  (New England Biolabs)
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    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with <t>λ-phosphatase</t> for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .
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    Image Search Results


    (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Journal: PLOS One

    Article Title: Mechanistic studies of PFKFB2 reveal a novel inhibitor of its kinase activity

    doi: 10.1371/journal.pone.0317167

    Figure Lengend Snippet: (A) Western blot analysis of cleared bacterial lysates of GST-PFKFB2 and mutants. Top: Total protein staining of cleared bacterial lysates over-expressing GST-PFKFB2 and mutants. Bottom: Western blots against GST and phosphorylated-PFKFB2 (Ser483) for GST-PFKFB2 and mutants. “Double mut” indicates protein containing both K174G and H259A. (B) Western blot analysis of GST-PFKFB2 expression, purification, and in vitro phosphorylation. Flow thru indicates proteins unbound to GSH beads. Column are proteins bound to the GSH beads. Column + Thrombin are proteins remaining bound to GSH beads after thrombin cleavage. Purified PFK2 lanes are proteins eluted from the GSH beads with thrombin cleavage. Purified PFK2 + ATP are proteins incubated with ATP alone. Purified PFK2 + ATP + PKA are proteins phosphorylated by PKA. (C) Western blot analysis of GST-PFKFB2 and mutant proteins showing they are phosphorylated (Ser483) upon extraction from bacteria ( left lanes ), lose phosphorylation after purification ( middle lanes ), but are re-phosphorylated upon treatment with PKA and ATP ( right lanes ). (D) Representative western blot against phosphorylated-PFKFB2 (Ser483) of bacterial samples (WT PFKFB2) treated/not treated with λ-phosphatase for indicated times before lysis. Equal amounts of purified PFKFB2 were treated and loaded into each lane. Quantitation of bacterial samples treated with λ-phosphatase. Data are shown as mean±SD. The original, unedited blots are included in .

    Article Snippet: Assay conditions were 100 µL sonicated sample, 10 µL MnCl 2 (10 mM NEB), 10 µL phosphatase buffer (10x PMP buffer, NEB) with or without 2 µL Lambda protein phosphatase (NEB, 400000 U/mL).

    Techniques: Western Blot, Staining, Expressing, Purification, In Vitro, Phospho-proteomics, Incubation, Mutagenesis, Extraction, Bacteria, Lysis, Quantitation Assay