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    Name:
    cAMP dependent Protein Kinase PKA
    Description:
    cAMP dependent Protein Kinase PKA 500 000 units
    Catalog Number:
    p6000l
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    500 000 units
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    Protein Kinases
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    Structured Review

    New England Biolabs pka
    cAMP dependent Protein Kinase PKA
    cAMP dependent Protein Kinase PKA 500 000 units
    https://www.bioz.com/result/pka/product/New England Biolabs
    Average 99 stars, based on 67 article reviews
    Price from $9.99 to $1999.99
    pka - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Nonsteroidal Anti-inflammatory Drugs Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL"

    Article Title: Nonsteroidal Anti-inflammatory Drugs Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL

    Journal: Journal of Virology

    doi: 10.1128/JVI.01018-18

    NSAIDs induce the dephosphorylation of CDC37 by the PGE 2 /cAMP/PKA pathway. (A) A549 cells were incubated with the NSAIDs for 48 h and then were treated with or without 5 μM PGE 2 for 24 h before harvest. The cells were subjected to Western blotting. (B) A549 cells were treated with different concentration of NSAIDs, and after 48 h the cells were harvested and lysed for detecting PGE 2 via ELISA. P values were determined by one-way ANOVA for comparison with the DMSO treatment ( n = 3 cultures). *, P
    Figure Legend Snippet: NSAIDs induce the dephosphorylation of CDC37 by the PGE 2 /cAMP/PKA pathway. (A) A549 cells were incubated with the NSAIDs for 48 h and then were treated with or without 5 μM PGE 2 for 24 h before harvest. The cells were subjected to Western blotting. (B) A549 cells were treated with different concentration of NSAIDs, and after 48 h the cells were harvested and lysed for detecting PGE 2 via ELISA. P values were determined by one-way ANOVA for comparison with the DMSO treatment ( n = 3 cultures). *, P

    Techniques Used: De-Phosphorylation Assay, Incubation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Schematic for NSAID-mediated AXL degradation. NSAIDs potently inhibit the COX-1/2 enzymatic activity and the production of PGE 2 . The signal through the EP2 receptor to cAMP consequently decreases. As a result, the protein kinase A (PKA) activity is inhibited. The S97 at the N-terminal domain of CDC37 is the specific target site for PKA. Its less phosphorylated status leads to a decreased interaction between CDC37 and HSP90. ZIKV entry cofactor AXL, as the client kinase, is improperly folded and eventually degraded by HSP70-CHIP-UPS.
    Figure Legend Snippet: Schematic for NSAID-mediated AXL degradation. NSAIDs potently inhibit the COX-1/2 enzymatic activity and the production of PGE 2 . The signal through the EP2 receptor to cAMP consequently decreases. As a result, the protein kinase A (PKA) activity is inhibited. The S97 at the N-terminal domain of CDC37 is the specific target site for PKA. Its less phosphorylated status leads to a decreased interaction between CDC37 and HSP90. ZIKV entry cofactor AXL, as the client kinase, is improperly folded and eventually degraded by HSP70-CHIP-UPS.

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation

    2) Product Images from "Protein Kinase A-mediated Serine 35 Phosphorylation Dissociates Histone H1.4 from Mitotic Chromosome *"

    Article Title: Protein Kinase A-mediated Serine 35 Phosphorylation Dissociates Histone H1.4 from Mitotic Chromosome *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.228064

    PKA mediates mitotic H1.4S35 phosphorylation in vivo . A , PKA overexpression induces H1.4S35 phosphorylation. 293T cells were transfected with indicated kinases for 24 h and subjected to Western blotting using indicated Ab. B , PKA inhibition reduces H1.4S35 phosphorylation. HeLa cells incubated with nocodazole (400 ng/ml) for 16 h were treated with kinase inhibitor H89 or KN-62 for 2 h, followed by Western blotting using Abs against the indicated proteins. C , PKA knockdown down-regulates H1.4S35 phosphorylation. HeLa cells depleted with PKAα and PKAβ by siRNA for 2 days were incubated with nocodazole for 16 h and subjected to Western blotting. D , expression of the dominant-negative PKA regulatory subunit inhibits H1.4S35 phosphorylation. HeLa cells with or without expressing the dominant-negative PKA regulatory subunit (F-PRKAR1α-DN) were incubated with nocodazole for 16 h and subjected to Western blotting using Abs against the indicated proteins. vec , vector; NT , mock-treated; TCL , total cell lysate.
    Figure Legend Snippet: PKA mediates mitotic H1.4S35 phosphorylation in vivo . A , PKA overexpression induces H1.4S35 phosphorylation. 293T cells were transfected with indicated kinases for 24 h and subjected to Western blotting using indicated Ab. B , PKA inhibition reduces H1.4S35 phosphorylation. HeLa cells incubated with nocodazole (400 ng/ml) for 16 h were treated with kinase inhibitor H89 or KN-62 for 2 h, followed by Western blotting using Abs against the indicated proteins. C , PKA knockdown down-regulates H1.4S35 phosphorylation. HeLa cells depleted with PKAα and PKAβ by siRNA for 2 days were incubated with nocodazole for 16 h and subjected to Western blotting. D , expression of the dominant-negative PKA regulatory subunit inhibits H1.4S35 phosphorylation. HeLa cells with or without expressing the dominant-negative PKA regulatory subunit (F-PRKAR1α-DN) were incubated with nocodazole for 16 h and subjected to Western blotting using Abs against the indicated proteins. vec , vector; NT , mock-treated; TCL , total cell lysate.

    Techniques Used: In Vivo, Over Expression, Transfection, Western Blot, Inhibition, Incubation, Expressing, Dominant Negative Mutation, Plasmid Preparation

    3) Product Images from "Phosphorylation of TIP3 Aquaporins during Phaseolus vulgaris Embryo Development"

    Article Title: Phosphorylation of TIP3 Aquaporins during Phaseolus vulgaris Embryo Development

    Journal: Cells

    doi: 10.3390/cells8111362

    Immunodetection of PvTIP3;1 phosphorylated at Ser7. ( A ) Dot blots of a phosphoserine-bearing protein mix (pSer) and of purified PvTIP3;1 treated with either lambda protein phosphatase (TIP/PPase) or protein kinase A catalytic subunit (TIP/PKA). Blots were immunolabeled with aTIPnt13C (lane 1), aTIPnt13C_S7P (lane 2), or phosphoserine (lane 3) antisera to test antibody specificity. Western immunoblots using ( B ) aTIPnt13C or ( C ) aTIPnt13C_S7P antisera. Blot lanes: IS, immature seed; MS, mature seed; DS, dry seed; 24, seed imbibed for 24 h; 48, seed imbibed for 48 h. Position of molecular weight markers (bars) with kDa values indicated to the right of panel C . ( D ) Graph showing ratio of aTIPnt13C_S7P to aTIPnt13C antisera labeling. Results displayed as the average of four experiments ± one standard deviation of the sample, normalized so that the labeling ratio is 1:1 for the dry seed fraction. Horizontal axis indicates seed fractions as in B and C .
    Figure Legend Snippet: Immunodetection of PvTIP3;1 phosphorylated at Ser7. ( A ) Dot blots of a phosphoserine-bearing protein mix (pSer) and of purified PvTIP3;1 treated with either lambda protein phosphatase (TIP/PPase) or protein kinase A catalytic subunit (TIP/PKA). Blots were immunolabeled with aTIPnt13C (lane 1), aTIPnt13C_S7P (lane 2), or phosphoserine (lane 3) antisera to test antibody specificity. Western immunoblots using ( B ) aTIPnt13C or ( C ) aTIPnt13C_S7P antisera. Blot lanes: IS, immature seed; MS, mature seed; DS, dry seed; 24, seed imbibed for 24 h; 48, seed imbibed for 48 h. Position of molecular weight markers (bars) with kDa values indicated to the right of panel C . ( D ) Graph showing ratio of aTIPnt13C_S7P to aTIPnt13C antisera labeling. Results displayed as the average of four experiments ± one standard deviation of the sample, normalized so that the labeling ratio is 1:1 for the dry seed fraction. Horizontal axis indicates seed fractions as in B and C .

    Techniques Used: Immunodetection, Purification, Immunolabeling, Western Blot, Mass Spectrometry, Molecular Weight, Labeling, Standard Deviation

    4) Product Images from "Discontinuous Actin Hexagon, a Protein Essential for Cortical Furrow Formation in Drosophila, Is Membrane Associated and Hyperphosphorylated"

    Article Title: Discontinuous Actin Hexagon, a Protein Essential for Cortical Furrow Formation in Drosophila, Is Membrane Associated and Hyperphosphorylated

    Journal: Molecular Biology of the Cell

    doi:

    DAH can be hyperphosphorylated by CKI. DAH protein was immunoprecipitated and the endogenous kinase associated with DAH was removed by 1 M KI (lanes 2–9). The DAH immunoprecipitates were then incubated with no kinases (−kin), or with casein kinase I (CK I), p34 cdc2 /cyclin B protein kinase (cdc2), casein kinase II (CK II), cAMP-dependent protein kinase (PKA), PKC, glycogen synthase kinase 3 (GSK-3), and MAPK for kinase reactions (lanes 3–9). These samples were prepared for gel electrophoresis, followed by analysis with silver staining (lower panel) and autoradiography (upper panel). Casein kinase I hyperphosphorylates DAH protein, whereas the other kinases fail to generate a similar phosphorylation pattern as the endogenous kinase.
    Figure Legend Snippet: DAH can be hyperphosphorylated by CKI. DAH protein was immunoprecipitated and the endogenous kinase associated with DAH was removed by 1 M KI (lanes 2–9). The DAH immunoprecipitates were then incubated with no kinases (−kin), or with casein kinase I (CK I), p34 cdc2 /cyclin B protein kinase (cdc2), casein kinase II (CK II), cAMP-dependent protein kinase (PKA), PKC, glycogen synthase kinase 3 (GSK-3), and MAPK for kinase reactions (lanes 3–9). These samples were prepared for gel electrophoresis, followed by analysis with silver staining (lower panel) and autoradiography (upper panel). Casein kinase I hyperphosphorylates DAH protein, whereas the other kinases fail to generate a similar phosphorylation pattern as the endogenous kinase.

    Techniques Used: Immunoprecipitation, Incubation, Nucleic Acid Electrophoresis, Silver Staining, Autoradiography

    5) Product Images from "Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling"

    Article Title: Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034936

    Potassium elevation induces cAMP production and increases astrocyte water permeability via PKA-dependent phosphorylation of AQP4. (A) 10mM potassium significantly increased WT AQP4 water permeability (*p
    Figure Legend Snippet: Potassium elevation induces cAMP production and increases astrocyte water permeability via PKA-dependent phosphorylation of AQP4. (A) 10mM potassium significantly increased WT AQP4 water permeability (*p

    Techniques Used: Permeability

    6) Product Images from "Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling"

    Article Title: Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling

    Journal: Nature medicine

    doi: 10.1038/nm.4011

    Activation of PKA stimulates hydrolyzing activity of the proteasome in slices-ex vivo and in vitro. (a–c) Immunoblot analysis and corresponding densitometric quantification of total and insoluble extracts for tau, pS214 tau epitope and GAPDH (a,c) and native PAGE analysis and quantification of 26S proteasome activity and level using antibody to the β5 subunit (b) in acute organotypic cortical slices from 3- to 4-month-old rTg4510 mice treated with vehicle, db-cAMP, rolipram or epoxomicin (Epox) alone or a combination of epoxomicin and db-cAMP or rolipram. (d–f) Hydrolysis rate of succinyl-Leu-Leu-Val-Tyr-amc (Suc-LLVY-amc) (d), 32P-labeled Ub5-DHFR substrate (e) and ATP (f) by purified 26S proteasomes from WT mice untreated (WT 26S), incubated with LMW tau aggregates and/or pre-incubated with PKA. (g) Immunoblot analysis of phosphorylated serine and threonine (pSer and pThr) epitopes of proteasome subunits by PKA in purified 26S proteasomes treated as in d–f. For a–c, two animals per experiment were analyzed in three biological replicates (n = 6 mice). For d–g, purified proteasomes from n = 6 brains from 3-month-old WT mice were used for each hydrolyzing assay and at least three independent experiments were performed. Error bars, mean ± s.e.m.; n.s., not significant; *P
    Figure Legend Snippet: Activation of PKA stimulates hydrolyzing activity of the proteasome in slices-ex vivo and in vitro. (a–c) Immunoblot analysis and corresponding densitometric quantification of total and insoluble extracts for tau, pS214 tau epitope and GAPDH (a,c) and native PAGE analysis and quantification of 26S proteasome activity and level using antibody to the β5 subunit (b) in acute organotypic cortical slices from 3- to 4-month-old rTg4510 mice treated with vehicle, db-cAMP, rolipram or epoxomicin (Epox) alone or a combination of epoxomicin and db-cAMP or rolipram. (d–f) Hydrolysis rate of succinyl-Leu-Leu-Val-Tyr-amc (Suc-LLVY-amc) (d), 32P-labeled Ub5-DHFR substrate (e) and ATP (f) by purified 26S proteasomes from WT mice untreated (WT 26S), incubated with LMW tau aggregates and/or pre-incubated with PKA. (g) Immunoblot analysis of phosphorylated serine and threonine (pSer and pThr) epitopes of proteasome subunits by PKA in purified 26S proteasomes treated as in d–f. For a–c, two animals per experiment were analyzed in three biological replicates (n = 6 mice). For d–g, purified proteasomes from n = 6 brains from 3-month-old WT mice were used for each hydrolyzing assay and at least three independent experiments were performed. Error bars, mean ± s.e.m.; n.s., not significant; *P

    Techniques Used: Activation Assay, Activity Assay, Ex Vivo, In Vitro, Clear Native PAGE, Mouse Assay, Labeling, Purification, Incubation

    7) Product Images from "Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain"

    Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.8.2716-2727.2002

    m-Calpain phosphorylation at ST369/370 by PKA limits proteolytic activity. Both His-tagged wild-type- and ST369AA hCANP-expressing cells were grown and made quiescent for 24 h. Cells were treated with dexamethasone (2 μM) for 18 h to induce expression of exogenous hCANP constructs. To determine whether m-calpain was a target of PKA, hCANP was purified by Ni-NTA affinity chromatography and dialyzed with 50 mM Tris-HCl (pH 7.4) for 24 h. (A) Purified hCANP was treated with constitutively active cAMP-dependent PKA catalytic subunit and [γ- 32 P]ATP for 15 min. The samples were analyzed by separation through an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was used for autoradiography and subsequently blotted with anti-m-calpain antibody (Santa Cruz). The results shown are representative of two independent experiments. (B) Purified hCANP was incubated with recombinant Tau protein with or without calcium (40 μM) in the presence or absence of ERK (300 U) for 10 min. Samples were analyzed by SDS-10% polyacrylamide gel electrophoresis and transferred to a nylon membrane (Immobilon-P). The transferred membrane was blotted with anti-Tau antibody (Zymed). Reduction in the size of Tau indicates calpain activity. The results shown are representative of two independent experiments. (C) Purified hCANP was incubated with MAP2 in the presence or absence of ERK (300 U) and PKA catalytic subunit (25 U) for 20 min. Samples were analyzed on an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was blotted with anti-MAP2 antibody (Sigma). Loss of cleavage products of 75 to 120 kDa indicated activity. The results shown are representative of two independent experiments.
    Figure Legend Snippet: m-Calpain phosphorylation at ST369/370 by PKA limits proteolytic activity. Both His-tagged wild-type- and ST369AA hCANP-expressing cells were grown and made quiescent for 24 h. Cells were treated with dexamethasone (2 μM) for 18 h to induce expression of exogenous hCANP constructs. To determine whether m-calpain was a target of PKA, hCANP was purified by Ni-NTA affinity chromatography and dialyzed with 50 mM Tris-HCl (pH 7.4) for 24 h. (A) Purified hCANP was treated with constitutively active cAMP-dependent PKA catalytic subunit and [γ- 32 P]ATP for 15 min. The samples were analyzed by separation through an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was used for autoradiography and subsequently blotted with anti-m-calpain antibody (Santa Cruz). The results shown are representative of two independent experiments. (B) Purified hCANP was incubated with recombinant Tau protein with or without calcium (40 μM) in the presence or absence of ERK (300 U) for 10 min. Samples were analyzed by SDS-10% polyacrylamide gel electrophoresis and transferred to a nylon membrane (Immobilon-P). The transferred membrane was blotted with anti-Tau antibody (Zymed). Reduction in the size of Tau indicates calpain activity. The results shown are representative of two independent experiments. (C) Purified hCANP was incubated with MAP2 in the presence or absence of ERK (300 U) and PKA catalytic subunit (25 U) for 20 min. Samples were analyzed on an SDS-10% polyacrylamide gel and transferred to a nylon membrane (Immobilon-P). The membrane was blotted with anti-MAP2 antibody (Sigma). Loss of cleavage products of 75 to 120 kDa indicated activity. The results shown are representative of two independent experiments.

    Techniques Used: Activity Assay, Expressing, Construct, Purification, Affinity Chromatography, Autoradiography, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis

    Related Articles

    Protease Inhibitor:

    Article Title: Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256
    Article Snippet: .. Forty microliters of 10 mM ATP (New England BioLabs), 1.5 μl of 100× EDTA-free Halt protease inhibitor (Pierce Biotechnology), and 60 μl of the phosphatase-treated sample (∼250-μg starting protein amount) were combined with PKA [catalytic subunit of cAMP-dependent protein kinase (New England BioLabs)], SGK [SGK I (CarnaBio, Natick, MA)], CAMK2 [CAMK IIδ (CarnaBio)], or vehicle, and water was added for a final reaction volume of 150 μl with a kinase concentration of 1.0 μM. .. For the CAMK2 samples, 0.5 mM CaCl2 and 0.03 μg/μl calmodulin (Sigma-Aldrich) were also added to the reaction mixture.

    Purification:

    Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain
    Article Snippet: .. Purified His-tagged m-calpains were incubated for 15 min at 30°C with an assay mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 200 μM [γ-32 P]ATP (final specific activity of 200 μCi/μmol), and 75 U of cAMP-dependent protein kinase catalytic subunit (New England Biolabs, Beverly, Mass.). .. The samples were separated by SDS-10% polyacrylamide gel electrophoresis, and then samples were transferred into an Immobilon-P (Millipore) PVDF membrane and exposed to X-ray film in a cassette for 72 h at −80°C.

    Article Title: Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling
    Article Snippet: .. Briefly, purified glutathione S-transferase (GST) fusion proteins, GST-APQ4 S111 or GST-APQ4 A111, were incubated with cAMP-dependent Protein Kinase (PKA), catalytic subunit (New England Biolabs) in PKA reaction buffer (New England Biolabs) containing 200 µM ATP, γ-32 pATP (0.2 µCi/lL), and Phosphatase Inhibitor Cocktail 2 (Sigma). ..

    Article Title: Discontinuous Actin Hexagon, a Protein Essential for Cortical Furrow Formation in Drosophila, Is Membrane Associated and Hyperphosphorylated
    Article Snippet: .. For exogenous kinase reactions, 5–20 U of purified kinases, casein kinase I, p34cdc2 /cyclin B protein kinase, casein kinase II, catalytic subunit of cAMP-dependent protein kinase, MAPK (Erk2), and glycogen synthase kinase 3, all of which were purchased from New England Biolabs , and the catalytic domain of PKC, which was generously provided by Drs. Y. Shi and P. Blackshear , were applied in a 30-μl reaction mixture. ..

    Concentration Assay:

    Article Title: Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256
    Article Snippet: .. Forty microliters of 10 mM ATP (New England BioLabs), 1.5 μl of 100× EDTA-free Halt protease inhibitor (Pierce Biotechnology), and 60 μl of the phosphatase-treated sample (∼250-μg starting protein amount) were combined with PKA [catalytic subunit of cAMP-dependent protein kinase (New England BioLabs)], SGK [SGK I (CarnaBio, Natick, MA)], CAMK2 [CAMK IIδ (CarnaBio)], or vehicle, and water was added for a final reaction volume of 150 μl with a kinase concentration of 1.0 μM. .. For the CAMK2 samples, 0.5 mM CaCl2 and 0.03 μg/μl calmodulin (Sigma-Aldrich) were also added to the reaction mixture.

    Incubation:

    Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain
    Article Snippet: .. Purified His-tagged m-calpains were incubated for 15 min at 30°C with an assay mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 200 μM [γ-32 P]ATP (final specific activity of 200 μCi/μmol), and 75 U of cAMP-dependent protein kinase catalytic subunit (New England Biolabs, Beverly, Mass.). .. The samples were separated by SDS-10% polyacrylamide gel electrophoresis, and then samples were transferred into an Immobilon-P (Millipore) PVDF membrane and exposed to X-ray film in a cassette for 72 h at −80°C.

    Article Title: Nonsteroidal Anti-inflammatory Drugs Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL
    Article Snippet: .. For each of the substrates, 100 ng was incubated with or without 1 μl (2,500 units) of the catalytic subunit of PKA (cAMP-dependent protein kinase [PKA]; New England BioLabs) in kinase buffer (50 mmol/liter Tris-HCl, pH 7.5, 10 mmol/liter MgCl2 , 2.5 mmol/liter ATP) in a total volume of 25 μl at 30°C for 1 h. The reactions were terminated by lowering the temperature to 0°C or by adding sample loading buffer. .. The proteins were then subjected to SDS-PAGE and detected by Western blotting using a phospho-(Ser/Thr) PKA substrate antibody.

    Article Title: Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling
    Article Snippet: .. Briefly, purified glutathione S-transferase (GST) fusion proteins, GST-APQ4 S111 or GST-APQ4 A111, were incubated with cAMP-dependent Protein Kinase (PKA), catalytic subunit (New England Biolabs) in PKA reaction buffer (New England Biolabs) containing 200 µM ATP, γ-32 pATP (0.2 µCi/lL), and Phosphatase Inhibitor Cocktail 2 (Sigma). ..

    Article Title: Protein Kinase A-mediated Serine 35 Phosphorylation Dissociates Histone H1.4 from Mitotic Chromosome *
    Article Snippet: .. Briefly, calf thymus H1 (14-155, Millipore) or core histones (10223565001, Roche Applied Science) was incubated with recombinant PKAα catalytic subunit (P6000, New England Biolabs) or recombinant Aurora B kinase (325901, Merck) in the presence of ATP at 37 °C for 1 h in kinase buffer (50 mm Tris-HCl and 10 mm MgCl2 ). .. Western blot was then applied using H1.4S35ph Ab or H3S10ph Ab.

    Activity Assay:

    Article Title: Activation of m-Calpain (Calpain II) by Epidermal Growth Factor Is Limited by Protein Kinase A Phosphorylation of m-Calpain
    Article Snippet: .. Purified His-tagged m-calpains were incubated for 15 min at 30°C with an assay mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 , 200 μM [γ-32 P]ATP (final specific activity of 200 μCi/μmol), and 75 U of cAMP-dependent protein kinase catalytic subunit (New England Biolabs, Beverly, Mass.). .. The samples were separated by SDS-10% polyacrylamide gel electrophoresis, and then samples were transferred into an Immobilon-P (Millipore) PVDF membrane and exposed to X-ray film in a cassette for 72 h at −80°C.

    Article Title: Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling
    Article Snippet: .. To confirm the effect on proteasome activity, 26S proteasomes (10 nM) were pre-treated with 400 U (1 uL) catalytic subunit of PKA (New England BioLabs #P6000S) for 60 min at 30 °C. .. Activated proteasomes were then incubated with recombinant tau forms (300 nM or 13.8 ng/µL) and 50 µM Suc-LLVY-amc peptide, and the fluorescence monitored as described above.

    Recombinant:

    Article Title: Protein Kinase A-mediated Serine 35 Phosphorylation Dissociates Histone H1.4 from Mitotic Chromosome *
    Article Snippet: .. Briefly, calf thymus H1 (14-155, Millipore) or core histones (10223565001, Roche Applied Science) was incubated with recombinant PKAα catalytic subunit (P6000, New England Biolabs) or recombinant Aurora B kinase (325901, Merck) in the presence of ATP at 37 °C for 1 h in kinase buffer (50 mm Tris-HCl and 10 mm MgCl2 ). .. Western blot was then applied using H1.4S35ph Ab or H3S10ph Ab.

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    New England Biolabs pka
    NSAIDs induce the dephosphorylation of CDC37 by the PGE 2 <t>/cAMP/PKA</t> pathway. (A) A549 cells were incubated with the NSAIDs for 48 h and then were treated with or without 5 μM PGE 2 for 24 h before harvest. The cells were subjected to Western blotting. (B) A549 cells were treated with different concentration of NSAIDs, and after 48 h the cells were harvested and lysed for detecting PGE 2 via ELISA. P values were determined by one-way ANOVA for comparison with the DMSO treatment ( n = 3 cultures). *, P
    Pka, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pka/product/New England Biolabs
    Average 99 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    pka - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    NSAIDs induce the dephosphorylation of CDC37 by the PGE 2 /cAMP/PKA pathway. (A) A549 cells were incubated with the NSAIDs for 48 h and then were treated with or without 5 μM PGE 2 for 24 h before harvest. The cells were subjected to Western blotting. (B) A549 cells were treated with different concentration of NSAIDs, and after 48 h the cells were harvested and lysed for detecting PGE 2 via ELISA. P values were determined by one-way ANOVA for comparison with the DMSO treatment ( n = 3 cultures). *, P

    Journal: Journal of Virology

    Article Title: Nonsteroidal Anti-inflammatory Drugs Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL

    doi: 10.1128/JVI.01018-18

    Figure Lengend Snippet: NSAIDs induce the dephosphorylation of CDC37 by the PGE 2 /cAMP/PKA pathway. (A) A549 cells were incubated with the NSAIDs for 48 h and then were treated with or without 5 μM PGE 2 for 24 h before harvest. The cells were subjected to Western blotting. (B) A549 cells were treated with different concentration of NSAIDs, and after 48 h the cells were harvested and lysed for detecting PGE 2 via ELISA. P values were determined by one-way ANOVA for comparison with the DMSO treatment ( n = 3 cultures). *, P

    Article Snippet: For each of the substrates, 100 ng was incubated with or without 1 μl (2,500 units) of the catalytic subunit of PKA (cAMP-dependent protein kinase [PKA]; New England BioLabs) in kinase buffer (50 mmol/liter Tris-HCl, pH 7.5, 10 mmol/liter MgCl2 , 2.5 mmol/liter ATP) in a total volume of 25 μl at 30°C for 1 h. The reactions were terminated by lowering the temperature to 0°C or by adding sample loading buffer.

    Techniques: De-Phosphorylation Assay, Incubation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Schematic for NSAID-mediated AXL degradation. NSAIDs potently inhibit the COX-1/2 enzymatic activity and the production of PGE 2 . The signal through the EP2 receptor to cAMP consequently decreases. As a result, the protein kinase A (PKA) activity is inhibited. The S97 at the N-terminal domain of CDC37 is the specific target site for PKA. Its less phosphorylated status leads to a decreased interaction between CDC37 and HSP90. ZIKV entry cofactor AXL, as the client kinase, is improperly folded and eventually degraded by HSP70-CHIP-UPS.

    Journal: Journal of Virology

    Article Title: Nonsteroidal Anti-inflammatory Drugs Potently Inhibit the Replication of Zika Viruses by Inducing the Degradation of AXL

    doi: 10.1128/JVI.01018-18

    Figure Lengend Snippet: Schematic for NSAID-mediated AXL degradation. NSAIDs potently inhibit the COX-1/2 enzymatic activity and the production of PGE 2 . The signal through the EP2 receptor to cAMP consequently decreases. As a result, the protein kinase A (PKA) activity is inhibited. The S97 at the N-terminal domain of CDC37 is the specific target site for PKA. Its less phosphorylated status leads to a decreased interaction between CDC37 and HSP90. ZIKV entry cofactor AXL, as the client kinase, is improperly folded and eventually degraded by HSP70-CHIP-UPS.

    Article Snippet: For each of the substrates, 100 ng was incubated with or without 1 μl (2,500 units) of the catalytic subunit of PKA (cAMP-dependent protein kinase [PKA]; New England BioLabs) in kinase buffer (50 mmol/liter Tris-HCl, pH 7.5, 10 mmol/liter MgCl2 , 2.5 mmol/liter ATP) in a total volume of 25 μl at 30°C for 1 h. The reactions were terminated by lowering the temperature to 0°C or by adding sample loading buffer.

    Techniques: Activity Assay, Chromatin Immunoprecipitation

    PKA mediates mitotic H1.4S35 phosphorylation in vivo . A , PKA overexpression induces H1.4S35 phosphorylation. 293T cells were transfected with indicated kinases for 24 h and subjected to Western blotting using indicated Ab. B , PKA inhibition reduces H1.4S35 phosphorylation. HeLa cells incubated with nocodazole (400 ng/ml) for 16 h were treated with kinase inhibitor H89 or KN-62 for 2 h, followed by Western blotting using Abs against the indicated proteins. C , PKA knockdown down-regulates H1.4S35 phosphorylation. HeLa cells depleted with PKAα and PKAβ by siRNA for 2 days were incubated with nocodazole for 16 h and subjected to Western blotting. D , expression of the dominant-negative PKA regulatory subunit inhibits H1.4S35 phosphorylation. HeLa cells with or without expressing the dominant-negative PKA regulatory subunit (F-PRKAR1α-DN) were incubated with nocodazole for 16 h and subjected to Western blotting using Abs against the indicated proteins. vec , vector; NT , mock-treated; TCL , total cell lysate.

    Journal: The Journal of Biological Chemistry

    Article Title: Protein Kinase A-mediated Serine 35 Phosphorylation Dissociates Histone H1.4 from Mitotic Chromosome *

    doi: 10.1074/jbc.M111.228064

    Figure Lengend Snippet: PKA mediates mitotic H1.4S35 phosphorylation in vivo . A , PKA overexpression induces H1.4S35 phosphorylation. 293T cells were transfected with indicated kinases for 24 h and subjected to Western blotting using indicated Ab. B , PKA inhibition reduces H1.4S35 phosphorylation. HeLa cells incubated with nocodazole (400 ng/ml) for 16 h were treated with kinase inhibitor H89 or KN-62 for 2 h, followed by Western blotting using Abs against the indicated proteins. C , PKA knockdown down-regulates H1.4S35 phosphorylation. HeLa cells depleted with PKAα and PKAβ by siRNA for 2 days were incubated with nocodazole for 16 h and subjected to Western blotting. D , expression of the dominant-negative PKA regulatory subunit inhibits H1.4S35 phosphorylation. HeLa cells with or without expressing the dominant-negative PKA regulatory subunit (F-PRKAR1α-DN) were incubated with nocodazole for 16 h and subjected to Western blotting using Abs against the indicated proteins. vec , vector; NT , mock-treated; TCL , total cell lysate.

    Article Snippet: Briefly, calf thymus H1 (14-155, Millipore) or core histones (10223565001, Roche Applied Science) was incubated with recombinant PKAα catalytic subunit (P6000, New England Biolabs) or recombinant Aurora B kinase (325901, Merck) in the presence of ATP at 37 °C for 1 h in kinase buffer (50 mm Tris-HCl and 10 mm MgCl2 ).

    Techniques: In Vivo, Over Expression, Transfection, Western Blot, Inhibition, Incubation, Expressing, Dominant Negative Mutation, Plasmid Preparation

    Immunodetection of PvTIP3;1 phosphorylated at Ser7. ( A ) Dot blots of a phosphoserine-bearing protein mix (pSer) and of purified PvTIP3;1 treated with either lambda protein phosphatase (TIP/PPase) or protein kinase A catalytic subunit (TIP/PKA). Blots were immunolabeled with aTIPnt13C (lane 1), aTIPnt13C_S7P (lane 2), or phosphoserine (lane 3) antisera to test antibody specificity. Western immunoblots using ( B ) aTIPnt13C or ( C ) aTIPnt13C_S7P antisera. Blot lanes: IS, immature seed; MS, mature seed; DS, dry seed; 24, seed imbibed for 24 h; 48, seed imbibed for 48 h. Position of molecular weight markers (bars) with kDa values indicated to the right of panel C . ( D ) Graph showing ratio of aTIPnt13C_S7P to aTIPnt13C antisera labeling. Results displayed as the average of four experiments ± one standard deviation of the sample, normalized so that the labeling ratio is 1:1 for the dry seed fraction. Horizontal axis indicates seed fractions as in B and C .

    Journal: Cells

    Article Title: Phosphorylation of TIP3 Aquaporins during Phaseolus vulgaris Embryo Development

    doi: 10.3390/cells8111362

    Figure Lengend Snippet: Immunodetection of PvTIP3;1 phosphorylated at Ser7. ( A ) Dot blots of a phosphoserine-bearing protein mix (pSer) and of purified PvTIP3;1 treated with either lambda protein phosphatase (TIP/PPase) or protein kinase A catalytic subunit (TIP/PKA). Blots were immunolabeled with aTIPnt13C (lane 1), aTIPnt13C_S7P (lane 2), or phosphoserine (lane 3) antisera to test antibody specificity. Western immunoblots using ( B ) aTIPnt13C or ( C ) aTIPnt13C_S7P antisera. Blot lanes: IS, immature seed; MS, mature seed; DS, dry seed; 24, seed imbibed for 24 h; 48, seed imbibed for 48 h. Position of molecular weight markers (bars) with kDa values indicated to the right of panel C . ( D ) Graph showing ratio of aTIPnt13C_S7P to aTIPnt13C antisera labeling. Results displayed as the average of four experiments ± one standard deviation of the sample, normalized so that the labeling ratio is 1:1 for the dry seed fraction. Horizontal axis indicates seed fractions as in B and C .

    Article Snippet: PvTIP3;1 treated with either the catalytic subunit of protein kinase A or with λ-protein phosphatase (New England BioLabs) served as positive and negative controls, respectively, for protein phosphorylation [ ].

    Techniques: Immunodetection, Purification, Immunolabeling, Western Blot, Mass Spectrometry, Molecular Weight, Labeling, Standard Deviation

    DAH can be hyperphosphorylated by CKI. DAH protein was immunoprecipitated and the endogenous kinase associated with DAH was removed by 1 M KI (lanes 2–9). The DAH immunoprecipitates were then incubated with no kinases (−kin), or with casein kinase I (CK I), p34 cdc2 /cyclin B protein kinase (cdc2), casein kinase II (CK II), cAMP-dependent protein kinase (PKA), PKC, glycogen synthase kinase 3 (GSK-3), and MAPK for kinase reactions (lanes 3–9). These samples were prepared for gel electrophoresis, followed by analysis with silver staining (lower panel) and autoradiography (upper panel). Casein kinase I hyperphosphorylates DAH protein, whereas the other kinases fail to generate a similar phosphorylation pattern as the endogenous kinase.

    Journal: Molecular Biology of the Cell

    Article Title: Discontinuous Actin Hexagon, a Protein Essential for Cortical Furrow Formation in Drosophila, Is Membrane Associated and Hyperphosphorylated

    doi:

    Figure Lengend Snippet: DAH can be hyperphosphorylated by CKI. DAH protein was immunoprecipitated and the endogenous kinase associated with DAH was removed by 1 M KI (lanes 2–9). The DAH immunoprecipitates were then incubated with no kinases (−kin), or with casein kinase I (CK I), p34 cdc2 /cyclin B protein kinase (cdc2), casein kinase II (CK II), cAMP-dependent protein kinase (PKA), PKC, glycogen synthase kinase 3 (GSK-3), and MAPK for kinase reactions (lanes 3–9). These samples were prepared for gel electrophoresis, followed by analysis with silver staining (lower panel) and autoradiography (upper panel). Casein kinase I hyperphosphorylates DAH protein, whereas the other kinases fail to generate a similar phosphorylation pattern as the endogenous kinase.

    Article Snippet: For exogenous kinase reactions, 5–20 U of purified kinases, casein kinase I, p34cdc2 /cyclin B protein kinase, casein kinase II, catalytic subunit of cAMP-dependent protein kinase, MAPK (Erk2), and glycogen synthase kinase 3, all of which were purchased from New England Biolabs , and the catalytic domain of PKC, which was generously provided by Drs. Y. Shi and P. Blackshear , were applied in a 30-μl reaction mixture.

    Techniques: Immunoprecipitation, Incubation, Nucleic Acid Electrophoresis, Silver Staining, Autoradiography