steady state atpase measurements atp hydrolysis  (Biomol GmbH)

 
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    Structured Review

    Biomol GmbH steady state atpase measurements atp hydrolysis
    Schematics of proposed <t>ATPase</t> cycle of the ATP-binding ( A ) and -hydrolysis ( B ) mutants and of the WT thermosome ( C ). The circles indicate the apo state, the squares the ATP-bound state and the hexagons the posthydrolysis closed state; the asterisk marks mutated subunits. For graphical clarity, only one of the four pairs of adjacent α–β subunits per ring is displayed. A ) Proposed rearrangements of the ATP-binding mutants. The scheme shows how the ATPase cycle in the unmodified subunit can be blocked either at the ATP-hydrolysis step (population of complex 1) or at the product release step (population of complex 2, boxed). B ) Proposed rearrangements of the ATP-hydrolysis mutants upon ATP binding. The β (tight) subunit has a higher ATPase activity than the α (weak), but both need to bind ATP to allow cycling of the adjacent subunit. For graphical simplicity, the scheme presents only the case in which ATP binds the β subunit first, although the opposite is, in principle, possible. C ) Proposed ATPase cycle for the WT Ta thermosome based on the asymmetric behavior and allostery displayed by the 2 subunits. The events are coordinated by the allosteric requirement for ATP to be bound to 1 subunit for the next one to release the hydrolysis products and be re-engaged in the cycle. Only the case in which ATP binding to 1 subunit is required for ADP and phosphate to be released from the neighboring one is reported (see Discussion for details).
    Steady State Atpase Measurements Atp Hydrolysis, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin"

    Article Title: Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin

    Journal: The FASEB Journal

    doi: 10.1096/fj.201701061R

    Schematics of proposed ATPase cycle of the ATP-binding ( A ) and -hydrolysis ( B ) mutants and of the WT thermosome ( C ). The circles indicate the apo state, the squares the ATP-bound state and the hexagons the posthydrolysis closed state; the asterisk marks mutated subunits. For graphical clarity, only one of the four pairs of adjacent α–β subunits per ring is displayed. A ) Proposed rearrangements of the ATP-binding mutants. The scheme shows how the ATPase cycle in the unmodified subunit can be blocked either at the ATP-hydrolysis step (population of complex 1) or at the product release step (population of complex 2, boxed). B ) Proposed rearrangements of the ATP-hydrolysis mutants upon ATP binding. The β (tight) subunit has a higher ATPase activity than the α (weak), but both need to bind ATP to allow cycling of the adjacent subunit. For graphical simplicity, the scheme presents only the case in which ATP binds the β subunit first, although the opposite is, in principle, possible. C ) Proposed ATPase cycle for the WT Ta thermosome based on the asymmetric behavior and allostery displayed by the 2 subunits. The events are coordinated by the allosteric requirement for ATP to be bound to 1 subunit for the next one to release the hydrolysis products and be re-engaged in the cycle. Only the case in which ATP binding to 1 subunit is required for ADP and phosphate to be released from the neighboring one is reported (see Discussion for details).
    Figure Legend Snippet: Schematics of proposed ATPase cycle of the ATP-binding ( A ) and -hydrolysis ( B ) mutants and of the WT thermosome ( C ). The circles indicate the apo state, the squares the ATP-bound state and the hexagons the posthydrolysis closed state; the asterisk marks mutated subunits. For graphical clarity, only one of the four pairs of adjacent α–β subunits per ring is displayed. A ) Proposed rearrangements of the ATP-binding mutants. The scheme shows how the ATPase cycle in the unmodified subunit can be blocked either at the ATP-hydrolysis step (population of complex 1) or at the product release step (population of complex 2, boxed). B ) Proposed rearrangements of the ATP-hydrolysis mutants upon ATP binding. The β (tight) subunit has a higher ATPase activity than the α (weak), but both need to bind ATP to allow cycling of the adjacent subunit. For graphical simplicity, the scheme presents only the case in which ATP binds the β subunit first, although the opposite is, in principle, possible. C ) Proposed ATPase cycle for the WT Ta thermosome based on the asymmetric behavior and allostery displayed by the 2 subunits. The events are coordinated by the allosteric requirement for ATP to be bound to 1 subunit for the next one to release the hydrolysis products and be re-engaged in the cycle. Only the case in which ATP binding to 1 subunit is required for ADP and phosphate to be released from the neighboring one is reported (see Discussion for details).

    Techniques Used: Binding Assay, Activity Assay

    ATPase and refolding activities of the WT thermosome and its mutants. A ) ATPase activity of the WT α 8 β 8 thermosome (open circles) and of the hydrolysis mutants D93Kβ α 8 β 8 (closed squares) and D94Aα α 8 β 8 (open squares) at 55°C as a function of ATP concentration. B ) ATPase activity of the α-only thermosome (α 16 ) at 55°C as a function of ATP concentration. Inset: linear dependence of the maximum hydrolytic rate of the 2 hydrolysis mutants on thermosome concentration. C ) Substrate refolding activity at 55°C, in the absence (empty symbols) and presence (filled symbols) of ATP, of the WT thermosome (red triangles) and of the hydrolysis mutants D93Kβ α 8 β 8 (green squares) and D94Aα α 8 β 8 (blue diamonds). The activity of the native substrate (T a RhaD) is indicated by the black closed circles, and the recovery in activity of unfolded Ta RhaD upon refolding in the absence of thermosome is illustrated by the black open circles. Results of such experiments in the presence of the homo-oligomeric complexes WT α 16 and D94Kα α 16 are reported in the inset. D ) Substrate refolding activity at 55°C in the absence (empty symbols) and presence (filled symbols) of ATP, of the WT α 8 β 8 thermosome (red triangles) and of the binding mutants T158Aβ α 8 β 8 (green squares) and T157Aα α 8 β 8 (blue diamonds). The refolding yields of the WT α 8 β 8 thermosome and all mutants are reported in Table 1 . Each data point reported is the mean of at least five independent experiments.
    Figure Legend Snippet: ATPase and refolding activities of the WT thermosome and its mutants. A ) ATPase activity of the WT α 8 β 8 thermosome (open circles) and of the hydrolysis mutants D93Kβ α 8 β 8 (closed squares) and D94Aα α 8 β 8 (open squares) at 55°C as a function of ATP concentration. B ) ATPase activity of the α-only thermosome (α 16 ) at 55°C as a function of ATP concentration. Inset: linear dependence of the maximum hydrolytic rate of the 2 hydrolysis mutants on thermosome concentration. C ) Substrate refolding activity at 55°C, in the absence (empty symbols) and presence (filled symbols) of ATP, of the WT thermosome (red triangles) and of the hydrolysis mutants D93Kβ α 8 β 8 (green squares) and D94Aα α 8 β 8 (blue diamonds). The activity of the native substrate (T a RhaD) is indicated by the black closed circles, and the recovery in activity of unfolded Ta RhaD upon refolding in the absence of thermosome is illustrated by the black open circles. Results of such experiments in the presence of the homo-oligomeric complexes WT α 16 and D94Kα α 16 are reported in the inset. D ) Substrate refolding activity at 55°C in the absence (empty symbols) and presence (filled symbols) of ATP, of the WT α 8 β 8 thermosome (red triangles) and of the binding mutants T158Aβ α 8 β 8 (green squares) and T157Aα α 8 β 8 (blue diamonds). The refolding yields of the WT α 8 β 8 thermosome and all mutants are reported in Table 1 . Each data point reported is the mean of at least five independent experiments.

    Techniques Used: Activity Assay, Concentration Assay, Binding Assay

    2) Product Images from "Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia *-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia * ♦"

    Article Title: Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia *-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.576157

    Transfection of HEK293 cells with cDNA encoding WT or R164H mutant SERCA1. A , cellular localization of WT and mutated SERCA1 proteins in HEK293 cells. Transfected cells were immunolabeled with monoclonal antibodies to SERCA1 and subsequently with antibodies to calreticulin, a molecular marker of ER. Cells were then incubated with TRITC-conjugated ( red fluorescence ) anti-mouse and FITC-conjugated ( green fluorescence ) anti-rabbit secondary antibodies. Simultaneous visualization of the two fluorochromes ( yellow signal ) shows that both WT and R164H mutant SERCA1 proteins are correctly targeted to ER, where co-localize with calreticulin. All panels are the same magnification ( scale bar , 10 μm). B , expression level analysis of WT and mutated SERCA1 proteins in transfected HEK293 cells. Total cell lysates from HEK293 cells transfected with either WT or R164H mutant SERCA1 cDNAs or with the empty vector were obtained by solubilization with 5% sodium deoxycholate. An equal quantity of total lysate from each sample was separated by SDS-PAGE and blotted onto nitrocellulose. The blot was incubated with antibodies specific for SERCA1 and the 43-kDa β-actin, used as loading control. In HEK293 cells transfected with the empty vector, SERCA1 antibody was unable to detect the protein. C , protein turnover of WT and mutated SERCA1. HEK293 cells transfected with WT ( circle ) or R164H mutant ( triangle ) SERCA1 cDNA were treated with cycloheximide ( CHX ), to inhibit protein synthesis, and then harvested at the indicated time points. An equal quantity of total cell lysate was separated by SDS-PAGE and blotted onto nitrocellulose. The blots were incubated with antibodies specific for SERCA1, and the 43-kDa β-actin and densitometric analysis was performed. Quantification data represent the ratio relative to time 0. The graph shows the average values (± S.D.) of at least three independent experiments.
    Figure Legend Snippet: Transfection of HEK293 cells with cDNA encoding WT or R164H mutant SERCA1. A , cellular localization of WT and mutated SERCA1 proteins in HEK293 cells. Transfected cells were immunolabeled with monoclonal antibodies to SERCA1 and subsequently with antibodies to calreticulin, a molecular marker of ER. Cells were then incubated with TRITC-conjugated ( red fluorescence ) anti-mouse and FITC-conjugated ( green fluorescence ) anti-rabbit secondary antibodies. Simultaneous visualization of the two fluorochromes ( yellow signal ) shows that both WT and R164H mutant SERCA1 proteins are correctly targeted to ER, where co-localize with calreticulin. All panels are the same magnification ( scale bar , 10 μm). B , expression level analysis of WT and mutated SERCA1 proteins in transfected HEK293 cells. Total cell lysates from HEK293 cells transfected with either WT or R164H mutant SERCA1 cDNAs or with the empty vector were obtained by solubilization with 5% sodium deoxycholate. An equal quantity of total lysate from each sample was separated by SDS-PAGE and blotted onto nitrocellulose. The blot was incubated with antibodies specific for SERCA1 and the 43-kDa β-actin, used as loading control. In HEK293 cells transfected with the empty vector, SERCA1 antibody was unable to detect the protein. C , protein turnover of WT and mutated SERCA1. HEK293 cells transfected with WT ( circle ) or R164H mutant ( triangle ) SERCA1 cDNA were treated with cycloheximide ( CHX ), to inhibit protein synthesis, and then harvested at the indicated time points. An equal quantity of total cell lysate was separated by SDS-PAGE and blotted onto nitrocellulose. The blots were incubated with antibodies specific for SERCA1, and the 43-kDa β-actin and densitometric analysis was performed. Quantification data represent the ratio relative to time 0. The graph shows the average values (± S.D.) of at least three independent experiments.

    Techniques Used: Transfection, Mutagenesis, Immunolabeling, Marker, Incubation, Fluorescence, Expressing, Plasmid Preparation, SDS Page

    Polyubiquitination of WT and R164H mutated SERCA1 proteins. HEK293 cells transfected with either WT or R164H mutant SERCA1 cDNA or the empty vector ( e.v ) were treated with the proteasome inhibitor MG132 (+) or its vehicle DMSO (−). A , solubilization of SERCA1 protein from transfected HEK293 cells. Transfected cells were lysed with radioimmunoprecipitation assay buffer (see “Experimental Procedures”) and centrifuged to obtain a soluble fraction ( lanes 1–5 ) and a pellet ( lanes 6–10 ). An equal quantity of protein or equal volume from solubilized supernatants and insoluble pellets, respectively, was separated by SDS-PAGE and probed with antibodies to SERCA1 and 43-kDa β-actin, used as loading control. B , ubiquitination status of WT and mutated SERCA1. From 200 μg of solubilized proteins (input for immunoprecipitation), SERCA1 was immunoprecipitated with the specific antibody. Immunocomplexes were resolved by SDS-PAGE. The ubiquitin-conjugated SERCA1 was tested with anti-ubiquitin ( Ub ) antibodies. The arrow indicates the IgG whole molecules used not fully denatured.
    Figure Legend Snippet: Polyubiquitination of WT and R164H mutated SERCA1 proteins. HEK293 cells transfected with either WT or R164H mutant SERCA1 cDNA or the empty vector ( e.v ) were treated with the proteasome inhibitor MG132 (+) or its vehicle DMSO (−). A , solubilization of SERCA1 protein from transfected HEK293 cells. Transfected cells were lysed with radioimmunoprecipitation assay buffer (see “Experimental Procedures”) and centrifuged to obtain a soluble fraction ( lanes 1–5 ) and a pellet ( lanes 6–10 ). An equal quantity of protein or equal volume from solubilized supernatants and insoluble pellets, respectively, was separated by SDS-PAGE and probed with antibodies to SERCA1 and 43-kDa β-actin, used as loading control. B , ubiquitination status of WT and mutated SERCA1. From 200 μg of solubilized proteins (input for immunoprecipitation), SERCA1 was immunoprecipitated with the specific antibody. Immunocomplexes were resolved by SDS-PAGE. The ubiquitin-conjugated SERCA1 was tested with anti-ubiquitin ( Ub ) antibodies. The arrow indicates the IgG whole molecules used not fully denatured.

    Techniques Used: Transfection, Mutagenesis, Plasmid Preparation, Radio Immunoprecipitation, SDS Page, Immunoprecipitation

    Ca 2+ -ATPase activity and expression levels of WT and R164H SERCA1 in microsomes isolated from HEK293 cells. ). A , Ca 2+ ). The values are normalized to that of WT SERCA1 (100%) and are the means ± S.D. of three independent experiments. B , microsomal fractions were separated by SDS-PAGE and blotted onto nitrocellulose. Expression of Ca 2+ -ATPase protein was detected by using an immunoblot procedure with antibody to SERCA1. The lower panel shows Ponceau Red staining of the membrane, used as loading control.
    Figure Legend Snippet: Ca 2+ -ATPase activity and expression levels of WT and R164H SERCA1 in microsomes isolated from HEK293 cells. ). A , Ca 2+ ). The values are normalized to that of WT SERCA1 (100%) and are the means ± S.D. of three independent experiments. B , microsomal fractions were separated by SDS-PAGE and blotted onto nitrocellulose. Expression of Ca 2+ -ATPase protein was detected by using an immunoblot procedure with antibody to SERCA1. The lower panel shows Ponceau Red staining of the membrane, used as loading control.

    Techniques Used: Activity Assay, Expressing, Isolation, SDS Page, Staining

    Proteasome inhibitor MG132 treatment allows the rescue of both expression levels and Ca 2+ -ATPase activity of R164H SERCA1 mutant. Bundles of fibers from muscle biopsy from PMT-affected Chianina cow, maintained under tissue culture conditions, were untreated ( untr. ), to obtain a crude microsomal fraction enriched in content of SR membranes, a soluble supernatant, and a myofibrillar fraction. A , immunodetection of SERCA1 in muscle subfractions. Microsomal fractions ( lanes 1–3 ), soluble supernatants ( lanes 4–6 ), and myofibrillar fractions ( lanes 7–9 ) were separated by SDS-PAGE and blotted into nitrocellulose (protein loading 5 μg/lane). Blots were incubated with antibody to SERCA1. The lower panels are Ponceau Red staining of the membrane, used as loading control. B , Ca 2+ -ATPase activity of SR microsomal fractions. The Ca 2+ -ATPase activity was determined by spectrophotometric assay at optimum p Ca ( p Ca 5) in the presence of Ca 2+ ionophore A23187. The values are the means of multiple determinations carried out on an individual preparation. Data are expressed as the percentage of values from the untreated sample: ***, p
    Figure Legend Snippet: Proteasome inhibitor MG132 treatment allows the rescue of both expression levels and Ca 2+ -ATPase activity of R164H SERCA1 mutant. Bundles of fibers from muscle biopsy from PMT-affected Chianina cow, maintained under tissue culture conditions, were untreated ( untr. ), to obtain a crude microsomal fraction enriched in content of SR membranes, a soluble supernatant, and a myofibrillar fraction. A , immunodetection of SERCA1 in muscle subfractions. Microsomal fractions ( lanes 1–3 ), soluble supernatants ( lanes 4–6 ), and myofibrillar fractions ( lanes 7–9 ) were separated by SDS-PAGE and blotted into nitrocellulose (protein loading 5 μg/lane). Blots were incubated with antibody to SERCA1. The lower panels are Ponceau Red staining of the membrane, used as loading control. B , Ca 2+ -ATPase activity of SR microsomal fractions. The Ca 2+ -ATPase activity was determined by spectrophotometric assay at optimum p Ca ( p Ca 5) in the presence of Ca 2+ ionophore A23187. The values are the means of multiple determinations carried out on an individual preparation. Data are expressed as the percentage of values from the untreated sample: ***, p

    Techniques Used: Expressing, Activity Assay, Mutagenesis, Immunodetection, SDS Page, Incubation, Staining, Spectrophotometric Assay

    Immunodetection of WT and R164H mutated SERCA1 proteins after incubation with proteasome inhibitors MG132, bortezomib, and lactacystin. HEK293 cells were transfected with WT, R164H mutant SERCA1 cDNAs, or the empty vector, when indicated. A , Western blot and densitometric analysis of SERCA1 protein. Cells were treated with the proteasome inhibitor MG132 (+) or its vehicle DMSO (−). An equal quantity of protein from total cell lysates, obtained by solubilization with 5% sodium deoxycholate, was separated by SDS-PAGE and subjected to immunoblot analysis with antibodies specific to SERCA1 and 43-kDa β-actin, used as loading control. The graph shows quantification of protein bands performed by densitometric analysis on Western blots. Data (means ± S.D. from six independent experiments) are reported as the percentage of WT SERCA1 expressed in HEK293 cells treated with the vehicle alone. ***, p ≤ 0.001. B , Western blot of SERCA1 protein. Cells were treated with the proteasome inhibitors bortezomib, MG132, and lactacystin or with their vehicle DMSO. An equal quantity of protein from total cell lysates obtained by solubilization with 5% sodium deoxycholate was separated by SDS-PAGE and subjected to immunoblot analysis with antibodies specific to SERCA1 and 43-kDa β-actin, used as loading control.
    Figure Legend Snippet: Immunodetection of WT and R164H mutated SERCA1 proteins after incubation with proteasome inhibitors MG132, bortezomib, and lactacystin. HEK293 cells were transfected with WT, R164H mutant SERCA1 cDNAs, or the empty vector, when indicated. A , Western blot and densitometric analysis of SERCA1 protein. Cells were treated with the proteasome inhibitor MG132 (+) or its vehicle DMSO (−). An equal quantity of protein from total cell lysates, obtained by solubilization with 5% sodium deoxycholate, was separated by SDS-PAGE and subjected to immunoblot analysis with antibodies specific to SERCA1 and 43-kDa β-actin, used as loading control. The graph shows quantification of protein bands performed by densitometric analysis on Western blots. Data (means ± S.D. from six independent experiments) are reported as the percentage of WT SERCA1 expressed in HEK293 cells treated with the vehicle alone. ***, p ≤ 0.001. B , Western blot of SERCA1 protein. Cells were treated with the proteasome inhibitors bortezomib, MG132, and lactacystin or with their vehicle DMSO. An equal quantity of protein from total cell lysates obtained by solubilization with 5% sodium deoxycholate was separated by SDS-PAGE and subjected to immunoblot analysis with antibodies specific to SERCA1 and 43-kDa β-actin, used as loading control.

    Techniques Used: Immunodetection, Incubation, Transfection, Mutagenesis, Plasmid Preparation, Western Blot, SDS Page

    3) Product Images from "G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase"

    Article Title: G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw349

    The presence of G4 structures lowers the ATPase activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P
    Figure Legend Snippet: The presence of G4 structures lowers the ATPase activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P

    Techniques Used: Activity Assay, Incubation

    4) Product Images from "Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation"

    Article Title: Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

    Journal: Scientific Reports

    doi: 10.1038/srep14498

    Effects of p62 on ARIP4 function. ( A ) Interaction between p62 and ARIP4 governs Ad4BP-mediated repression activity of the target promoter. HEK293 cells were transiently transfected with the luciferase reporter gene ( StAR -Luc), Ad4BP/SF-1, and the indicated amounts (ng) of ARIP4 wild-type (WT) or ARIP4 mutants carrying an internal deletion (dSID1 or dSID2). The relative luciferase activity is shown: the amount of Ad4BP/SF-1 activation without ARIP4 expression was set at 100%. The lower panels represent ARIP4 wild-type and mutant protein levels, determined using an anti-FLAG M2 antibody. The control for the efficiency of transfection (Ad4BP/SF-1) and the loading control (GAPDH) are shown in separate panels. ( B ) The ATPase activity of ARIP4 does not modulate the p62-binding domain, SID. FLAG-tagged ARIP4 was expressed in HEK293 cells and purified with anti-FLAG M2 agarose as ARIP4 complexes. Either wild-type, dSID1, dSID2 or the ATPase mutant (K311A) of ARIP4 (100 ng) was incubated with dsDNA (1 μg). The relative ATPase activity is shown: the ATPase activity of ARIP4 WT was set at 100%, and the data are represented as the mean ± SD (upper panels; n = 3). ARIP4 WT and mutant protein levels were determined using western blot analysis with anti-ARIP4 antibody (lower panels). ( C ) Protein levels of ARIP4 and p62 decreased under nutrient starvation conditions. Wild-type and p62 KO MEF cells were cultured in starvation medium lacking amino acids and serum for 2 h, with or without the autophagy inhibitor, BafA1. Cell lysates were analysed by immunoblotting using the indicated antibodies. ( D ) Protein levels of ectopically expressed ARIP4 decreased under nutrient starvation conditions in U2OS cells. FLAG-tagged ARIP4 (WT) and their SID deletion mutants (dSID1 and dSID2) were expressed in U2OS cells. These cells were cultured in starvation medium lacking amino acids and serum for 6 h. Cell lysates were analysed by immunoblotting using the indicated antibodies.
    Figure Legend Snippet: Effects of p62 on ARIP4 function. ( A ) Interaction between p62 and ARIP4 governs Ad4BP-mediated repression activity of the target promoter. HEK293 cells were transiently transfected with the luciferase reporter gene ( StAR -Luc), Ad4BP/SF-1, and the indicated amounts (ng) of ARIP4 wild-type (WT) or ARIP4 mutants carrying an internal deletion (dSID1 or dSID2). The relative luciferase activity is shown: the amount of Ad4BP/SF-1 activation without ARIP4 expression was set at 100%. The lower panels represent ARIP4 wild-type and mutant protein levels, determined using an anti-FLAG M2 antibody. The control for the efficiency of transfection (Ad4BP/SF-1) and the loading control (GAPDH) are shown in separate panels. ( B ) The ATPase activity of ARIP4 does not modulate the p62-binding domain, SID. FLAG-tagged ARIP4 was expressed in HEK293 cells and purified with anti-FLAG M2 agarose as ARIP4 complexes. Either wild-type, dSID1, dSID2 or the ATPase mutant (K311A) of ARIP4 (100 ng) was incubated with dsDNA (1 μg). The relative ATPase activity is shown: the ATPase activity of ARIP4 WT was set at 100%, and the data are represented as the mean ± SD (upper panels; n = 3). ARIP4 WT and mutant protein levels were determined using western blot analysis with anti-ARIP4 antibody (lower panels). ( C ) Protein levels of ARIP4 and p62 decreased under nutrient starvation conditions. Wild-type and p62 KO MEF cells were cultured in starvation medium lacking amino acids and serum for 2 h, with or without the autophagy inhibitor, BafA1. Cell lysates were analysed by immunoblotting using the indicated antibodies. ( D ) Protein levels of ectopically expressed ARIP4 decreased under nutrient starvation conditions in U2OS cells. FLAG-tagged ARIP4 (WT) and their SID deletion mutants (dSID1 and dSID2) were expressed in U2OS cells. These cells were cultured in starvation medium lacking amino acids and serum for 6 h. Cell lysates were analysed by immunoblotting using the indicated antibodies.

    Techniques Used: Activity Assay, Transfection, Luciferase, Activation Assay, Expressing, Mutagenesis, Binding Assay, Purification, Incubation, Western Blot, Cell Culture

    5) Product Images from "Protein Carbonylation of an Amino Acid Residue of the Na/K‐ATPase α1 Subunit Determines Na/K‐ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells"

    Article Title: Protein Carbonylation of an Amino Acid Residue of the Na/K‐ATPase α1 Subunit Determines Na/K‐ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    doi: 10.1161/JAHA.116.003675

    P224A mutation prevents ouabain‐induced Na/K‐ATPase endocytosis. P224A mutation prevents ouabain (10 μmol/L, 1 hour)‐stimulated accumulation of α1 and β1 subunit in early endosome fractions. A representative Western blot and quantitative analysis were shown. n=4, ** P
    Figure Legend Snippet: P224A mutation prevents ouabain‐induced Na/K‐ATPase endocytosis. P224A mutation prevents ouabain (10 μmol/L, 1 hour)‐stimulated accumulation of α1 and β1 subunit in early endosome fractions. A representative Western blot and quantitative analysis were shown. n=4, ** P

    Techniques Used: Mutagenesis, Western Blot

    Expression of Na/K‐ATPase in P224A mutation. A and B, P224A mutant cells express mutated rat α1 and β1 subunits. Expression of the rat α1 Na/K‐ATPase was determined with polyclonal rat α1‐specific antibody (anti‐NASE) (n=4) and the total α1 was determined with monoclonal anti‐α1 antibody (clone α6F) (n=3). Expression of endogenous pig β1 subunit (glycosylated) was determined with monoclonal anti‐β1 antibody (clone C464.8) (n=4) and the blots were optimized to show possible difference. A representative Western blot and quantitative analysis were shown. Quantitative analysis (bar graph) showed the relative expression of α1 and β1 subunits to control wild‐type AAC‐19 (for rat α1 and β1), and LLC‐PK1 (for total α1) cells. ** P
    Figure Legend Snippet: Expression of Na/K‐ATPase in P224A mutation. A and B, P224A mutant cells express mutated rat α1 and β1 subunits. Expression of the rat α1 Na/K‐ATPase was determined with polyclonal rat α1‐specific antibody (anti‐NASE) (n=4) and the total α1 was determined with monoclonal anti‐α1 antibody (clone α6F) (n=3). Expression of endogenous pig β1 subunit (glycosylated) was determined with monoclonal anti‐β1 antibody (clone C464.8) (n=4) and the blots were optimized to show possible difference. A representative Western blot and quantitative analysis were shown. Quantitative analysis (bar graph) showed the relative expression of α1 and β1 subunits to control wild‐type AAC‐19 (for rat α1 and β1), and LLC‐PK1 (for total α1) cells. ** P

    Techniques Used: Expressing, Mutagenesis, Western Blot

    Illustration of the 3‐dimensional structure of the Na/K‐ATPase α1 subunit in E1P state (left panel), E2P state (right panel), and Pro222 (middle panel). From upper to lower images, Pro222, carbonylated 222, and Ala222 in Pro/Ala mutation in E1P state.
    Figure Legend Snippet: Illustration of the 3‐dimensional structure of the Na/K‐ATPase α1 subunit in E1P state (left panel), E2P state (right panel), and Pro222 (middle panel). From upper to lower images, Pro222, carbonylated 222, and Ala222 in Pro/Ala mutation in E1P state.

    Techniques Used: Mutagenesis

    P224A mutation prevents ouabain‐stimulated protein carbonylation and Na/K‐ATPase signaling. Ouabain (Oua, 10 μmol/L, 1 hour)‐stimulated protein (A) protein carbonylation (n=5 for AAC‐19, n=6 for P224A, and n=4 for A416P cells), (B) activation of c‐Src (n=4), and (C) activation of ERK1/2 (n=5 for AAC‐19, and n=4 for P224A and A416P cells) in control AAC‐19 and mutant A416P cells, but not in P224A cells. Ponceau S staining served as loading control for carbonylation. Activation of c‐Src and ERK1/2 was expressed as the ratio of phosphorylated c‐Src (p‐Src) vs total c‐Src (t‐Src) and the ratio of phosphorylated ERK1/2 (p‐ERK) vs total ERK1/2 (t‐ERK), respectively. ** P
    Figure Legend Snippet: P224A mutation prevents ouabain‐stimulated protein carbonylation and Na/K‐ATPase signaling. Ouabain (Oua, 10 μmol/L, 1 hour)‐stimulated protein (A) protein carbonylation (n=5 for AAC‐19, n=6 for P224A, and n=4 for A416P cells), (B) activation of c‐Src (n=4), and (C) activation of ERK1/2 (n=5 for AAC‐19, and n=4 for P224A and A416P cells) in control AAC‐19 and mutant A416P cells, but not in P224A cells. Ponceau S staining served as loading control for carbonylation. Activation of c‐Src and ERK1/2 was expressed as the ratio of phosphorylated c‐Src (p‐Src) vs total c‐Src (t‐Src) and the ratio of phosphorylated ERK1/2 (p‐ERK) vs total ERK1/2 (t‐ERK), respectively. ** P

    Techniques Used: Mutagenesis, Activation Assay, Staining

    6) Product Images from "G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase"

    Article Title: G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw349

    The presence of G4 structures lowers the ATPase activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P
    Figure Legend Snippet: The presence of G4 structures lowers the ATPase activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P

    Techniques Used: Activity Assay, Incubation

    7) Product Images from "Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated Nuclear Receptor Ad4BP/SF-1"

    Article Title: Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated Nuclear Receptor Ad4BP/SF-1

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E08-12-1247

    The ATPase activity of ARIP4 is enhanced in the presence of sumoylated Ad4BP/SF-1 and dsDNA containing an Ad4 site. (A) Purity of the ARIP4, Ad4BP/SF-1, and the in vitro sumoylation products used in the ATPase assay were shown. FLAG-tagged ARIP4 and Ad4BP/SF-1
    Figure Legend Snippet: The ATPase activity of ARIP4 is enhanced in the presence of sumoylated Ad4BP/SF-1 and dsDNA containing an Ad4 site. (A) Purity of the ARIP4, Ad4BP/SF-1, and the in vitro sumoylation products used in the ATPase assay were shown. FLAG-tagged ARIP4 and Ad4BP/SF-1

    Techniques Used: Activity Assay, In Vitro, ATPase Assay

    8) Product Images from "Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus"

    Article Title: Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.02204-18

    ClpC1 ATPase activity (A) and proteolytic activity of the ClpC1/P1/P2 complex (B) in response to ECU and RUFI treatment. This experiment was carried out in triplicate.
    Figure Legend Snippet: ClpC1 ATPase activity (A) and proteolytic activity of the ClpC1/P1/P2 complex (B) in response to ECU and RUFI treatment. This experiment was carried out in triplicate.

    Techniques Used: Activity Assay

    Related Articles

    Immunostaining:

    Article Title: Isoform-Specific Up-Regulation of Plasma Membrane Ca2+ATPase Expression During Colon and Gastric Cancer Cell Differentiation
    Article Snippet: .. Immunostaining for the detection of Na+ /K+ -ATPase, carcinoembryonic antigen (CEA) and of dipeptidyl-peptidase IV (DPP-IV) was performed using the anti-Na+ /K+ -ATPase, clone no.: XVIF9-G10 (BIOMOL), the anti-CEA, clone no.: C6G9 (Sigma-Aldrich) and the anti-DPP-IV, clone no.: HBB 3/775/42 monoclonal antibodies at 1:1000, 1:1500 and 1:800 dilutions, respectively [ , ]. .. Antibody binding on PVDF-membranes was detected using the enhanced chemiluminescence (ECL) Western-blot reagents from Amersham International, UK.

    Modification:

    Article Title: Intraring allostery controls the function and assembly of a hetero-oligomeric class II chaperonin
    Article Snippet: .. Steady-state ATPase measurements ATP hydrolysis was measured using a modification of the classic malachite green reagent ( ) (Biomol Green; Enzo Life Sciences, Farmingdale, NY, USA) allowing for thermostable and reliable measurements of inorganic phosphate release at 55°C. ..

    Incubation:

    Article Title: Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough
    Article Snippet: .. Wholemounts were covered in blocking solution (10% goat serum, 1% BSA, 0.5% Tween 20 in PBS) for 1 h and then incubated overnight at 37°C with (1) a polyclonal rabbit antibody to the pan-neuronal marker, protein gene product (PGP) 9.5 (1:400, Millipore Bioscience Research Reagents); (2) a monoclonal rat antibody to substance P (1:200, Millipore Bioscience Research Reagents); or (3) a monoclonal mouse antibody to the α3 subunit of Na+ -K+ ATPase (clone XVIF9G10, 1:600, Biomol International, Plymouth Meeting, PA). .. Staining was detected with rhodamine- or fluorescein-conjugated goat anti-rabbit/rat/mouse IgG (1:200 dilution, Invitrogen).

    Article Title: Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia *-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia * ♦
    Article Snippet: .. The cells were permeabilized in 0.5% Triton X-100 in PBS and incubated with the following primary antibodies: mouse monoclonal antibody to SERCA1 (Biomol) (recognizing an epitope between amino acid residues 506 and C terminus of rabbit fast-twitch SR Ca2+ -ATPase) (dilution 1:500); and rabbit polyclonal antibody to calreticulin (Affinity Bioreagents) (dilution 1:100). .. Cells were then incubated with the appropriate secondary antibody conjugated with TRITC or FITC (Dako).

    Activity Assay:

    Article Title: G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase
    Article Snippet: .. The presence of G4 structures strongly inhibits the ATPase activity of nPfh1 The ATPase activity of nPfh1 was measured by using the BIOMOL Green reagent assay that quantifies the amount of free phosphate released by the protein. ..

    Article Title: Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation
    Article Snippet: .. The ATPase activity of ARIP4 was determined using Biomol Green reagent (Biomol Research Laboratories) according to the manufacturer’s protocol. .. Purified FLAG-tagged ARIP4 was incubated in a 50 μL reaction containing 20 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM MgCl2 and 200 μM ATP in the presence of dsDNA. pBlueScript SK(+) was used as the dsDNA.

    Article Title: Protein Carbonylation of an Amino Acid Residue of the Na/K‐ATPase α1 Subunit Determines Na/K‐ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells
    Article Snippet: .. Enzymatic and Ion‐Exchange Activity Assays The enzymatic activity of the Na/K‐ATPase was performed by using BIOMOL GREEN Reagent (Enzo Life Science) as described in . .. Briefly, cells were homogenized, briefly sonicated, and centrifuged (800g for 10 minutes) in ice‐cold buffer A (150 mmol/L sucrose, 5 mmol/L HEPES, 4 mmol/L EGTA, 0.8 mmol/L dithiothreitol, pH 7.4).

    Blocking Assay:

    Article Title: Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough
    Article Snippet: .. Wholemounts were covered in blocking solution (10% goat serum, 1% BSA, 0.5% Tween 20 in PBS) for 1 h and then incubated overnight at 37°C with (1) a polyclonal rabbit antibody to the pan-neuronal marker, protein gene product (PGP) 9.5 (1:400, Millipore Bioscience Research Reagents); (2) a monoclonal rat antibody to substance P (1:200, Millipore Bioscience Research Reagents); or (3) a monoclonal mouse antibody to the α3 subunit of Na+ -K+ ATPase (clone XVIF9G10, 1:600, Biomol International, Plymouth Meeting, PA). .. Staining was detected with rhodamine- or fluorescein-conjugated goat anti-rabbit/rat/mouse IgG (1:200 dilution, Invitrogen).

    Marker:

    Article Title: Selective Expression of a Sodium Pump Isozyme by Cough Receptors and Evidence for Its Essential Role in Regulating Cough
    Article Snippet: .. Wholemounts were covered in blocking solution (10% goat serum, 1% BSA, 0.5% Tween 20 in PBS) for 1 h and then incubated overnight at 37°C with (1) a polyclonal rabbit antibody to the pan-neuronal marker, protein gene product (PGP) 9.5 (1:400, Millipore Bioscience Research Reagents); (2) a monoclonal rat antibody to substance P (1:200, Millipore Bioscience Research Reagents); or (3) a monoclonal mouse antibody to the α3 subunit of Na+ -K+ ATPase (clone XVIF9G10, 1:600, Biomol International, Plymouth Meeting, PA). .. Staining was detected with rhodamine- or fluorescein-conjugated goat anti-rabbit/rat/mouse IgG (1:200 dilution, Invitrogen).

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    Biomol GmbH atpase activity
    The presence of <t>G4</t> structures lowers the <t>ATPase</t> activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P
    Atpase Activity, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The presence of G4 structures lowers the ATPase activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P

    Journal: Nucleic Acids Research

    Article Title: G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structures in vitro and are unwound by the Pfh1 DNA helicase

    doi: 10.1093/nar/gkw349

    Figure Lengend Snippet: The presence of G4 structures lowers the ATPase activity of nPfh1. ( A ) Measurements of the amount of ATP hydrolyzed by 1, 2 or 10 nM nPfh1 after 10 min incubation in the presence of 5 nM single-stranded oligonucleotide (57 nt), rDNA or rDNA-m4 folded in 100 mM NaCl. ( B ) The amount of ATP hydrolyzed by 2 nM nPfh1 after 20 min incubation the presence of 100 nM rDNA, rDNA-m4, telomere or telomere-m4 oligonucleotides folded in 1 M KCl, in the absence or presence of 500 nM Phen-DC 3 . Data in A and B are shown as the average of three samples and error bars represent standard deviations. Significant differences ( P

    Article Snippet: The presence of G4 structures strongly inhibits the ATPase activity of nPfh1 The ATPase activity of nPfh1 was measured by using the BIOMOL Green reagent assay that quantifies the amount of free phosphate released by the protein.

    Techniques: Activity Assay, Incubation

    Effects of p62 on ARIP4 function. ( A ) Interaction between p62 and ARIP4 governs Ad4BP-mediated repression activity of the target promoter. HEK293 cells were transiently transfected with the luciferase reporter gene ( StAR -Luc), Ad4BP/SF-1, and the indicated amounts (ng) of ARIP4 wild-type (WT) or ARIP4 mutants carrying an internal deletion (dSID1 or dSID2). The relative luciferase activity is shown: the amount of Ad4BP/SF-1 activation without ARIP4 expression was set at 100%. The lower panels represent ARIP4 wild-type and mutant protein levels, determined using an anti-FLAG M2 antibody. The control for the efficiency of transfection (Ad4BP/SF-1) and the loading control (GAPDH) are shown in separate panels. ( B ) The ATPase activity of ARIP4 does not modulate the p62-binding domain, SID. FLAG-tagged ARIP4 was expressed in HEK293 cells and purified with anti-FLAG M2 agarose as ARIP4 complexes. Either wild-type, dSID1, dSID2 or the ATPase mutant (K311A) of ARIP4 (100 ng) was incubated with dsDNA (1 μg). The relative ATPase activity is shown: the ATPase activity of ARIP4 WT was set at 100%, and the data are represented as the mean ± SD (upper panels; n = 3). ARIP4 WT and mutant protein levels were determined using western blot analysis with anti-ARIP4 antibody (lower panels). ( C ) Protein levels of ARIP4 and p62 decreased under nutrient starvation conditions. Wild-type and p62 KO MEF cells were cultured in starvation medium lacking amino acids and serum for 2 h, with or without the autophagy inhibitor, BafA1. Cell lysates were analysed by immunoblotting using the indicated antibodies. ( D ) Protein levels of ectopically expressed ARIP4 decreased under nutrient starvation conditions in U2OS cells. FLAG-tagged ARIP4 (WT) and their SID deletion mutants (dSID1 and dSID2) were expressed in U2OS cells. These cells were cultured in starvation medium lacking amino acids and serum for 6 h. Cell lysates were analysed by immunoblotting using the indicated antibodies.

    Journal: Scientific Reports

    Article Title: Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation

    doi: 10.1038/srep14498

    Figure Lengend Snippet: Effects of p62 on ARIP4 function. ( A ) Interaction between p62 and ARIP4 governs Ad4BP-mediated repression activity of the target promoter. HEK293 cells were transiently transfected with the luciferase reporter gene ( StAR -Luc), Ad4BP/SF-1, and the indicated amounts (ng) of ARIP4 wild-type (WT) or ARIP4 mutants carrying an internal deletion (dSID1 or dSID2). The relative luciferase activity is shown: the amount of Ad4BP/SF-1 activation without ARIP4 expression was set at 100%. The lower panels represent ARIP4 wild-type and mutant protein levels, determined using an anti-FLAG M2 antibody. The control for the efficiency of transfection (Ad4BP/SF-1) and the loading control (GAPDH) are shown in separate panels. ( B ) The ATPase activity of ARIP4 does not modulate the p62-binding domain, SID. FLAG-tagged ARIP4 was expressed in HEK293 cells and purified with anti-FLAG M2 agarose as ARIP4 complexes. Either wild-type, dSID1, dSID2 or the ATPase mutant (K311A) of ARIP4 (100 ng) was incubated with dsDNA (1 μg). The relative ATPase activity is shown: the ATPase activity of ARIP4 WT was set at 100%, and the data are represented as the mean ± SD (upper panels; n = 3). ARIP4 WT and mutant protein levels were determined using western blot analysis with anti-ARIP4 antibody (lower panels). ( C ) Protein levels of ARIP4 and p62 decreased under nutrient starvation conditions. Wild-type and p62 KO MEF cells were cultured in starvation medium lacking amino acids and serum for 2 h, with or without the autophagy inhibitor, BafA1. Cell lysates were analysed by immunoblotting using the indicated antibodies. ( D ) Protein levels of ectopically expressed ARIP4 decreased under nutrient starvation conditions in U2OS cells. FLAG-tagged ARIP4 (WT) and their SID deletion mutants (dSID1 and dSID2) were expressed in U2OS cells. These cells were cultured in starvation medium lacking amino acids and serum for 6 h. Cell lysates were analysed by immunoblotting using the indicated antibodies.

    Article Snippet: The ATPase activity of ARIP4 was determined using Biomol Green reagent (Biomol Research Laboratories) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Transfection, Luciferase, Activation Assay, Expressing, Mutagenesis, Binding Assay, Purification, Incubation, Western Blot, Cell Culture

    The ATPase activity of ARIP4 is enhanced in the presence of sumoylated Ad4BP/SF-1 and dsDNA containing an Ad4 site. (A) Purity of the ARIP4, Ad4BP/SF-1, and the in vitro sumoylation products used in the ATPase assay were shown. FLAG-tagged ARIP4 and Ad4BP/SF-1

    Journal: Molecular Biology of the Cell

    Article Title: Transcriptional Suppression by Transient Recruitment of ARIP4 to Sumoylated Nuclear Receptor Ad4BP/SF-1

    doi: 10.1091/mbc.E08-12-1247

    Figure Lengend Snippet: The ATPase activity of ARIP4 is enhanced in the presence of sumoylated Ad4BP/SF-1 and dsDNA containing an Ad4 site. (A) Purity of the ARIP4, Ad4BP/SF-1, and the in vitro sumoylation products used in the ATPase assay were shown. FLAG-tagged ARIP4 and Ad4BP/SF-1

    Article Snippet: The ATPase activity of ARIP4 was determined using BIOMOL GREEN reagent (BIOMOL Research Laboratories, Plymouth Meeting, PA) according to the manufacturer's protocol.

    Techniques: Activity Assay, In Vitro, ATPase Assay