Structured Review

Cytoskeleton Inc atpases
Effect of actin on the Ca 2+ <t>-ATPase</t> activity and phosphorylated intermediates in isolated human erythrocyte PMCA during early stages of actin polymerization. A , Ca 2+ -ATPase activity was measured in PMCA reconstituted in DMPC/C 12 E 10 -mixed micelles in the
Atpases, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/atpases/product/Cytoskeleton Inc
Average 89 stars, based on 1 article reviews
Price from $9.99 to $1999.99
atpases - by Bioz Stars, 2020-05
89/100 stars

Images

1) Product Images from "Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction *"

Article Title: Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.470542

Effect of actin on the Ca 2+ -ATPase activity and phosphorylated intermediates in isolated human erythrocyte PMCA during early stages of actin polymerization. A , Ca 2+ -ATPase activity was measured in PMCA reconstituted in DMPC/C 12 E 10 -mixed micelles in the
Figure Legend Snippet: Effect of actin on the Ca 2+ -ATPase activity and phosphorylated intermediates in isolated human erythrocyte PMCA during early stages of actin polymerization. A , Ca 2+ -ATPase activity was measured in PMCA reconstituted in DMPC/C 12 E 10 -mixed micelles in the

Techniques Used: Activity Assay, Isolation

[Ca 2+ ] dependence of G-actin effect on human erythrocyte PMCA Ca 2+ -ATPase activity. Ca 2+ -ATPase activity was measured at different [Ca 2+ ] in the presence of the following: 100 n m calmodulin (▴) as a positive control for PMCA activation; 5 μ
Figure Legend Snippet: [Ca 2+ ] dependence of G-actin effect on human erythrocyte PMCA Ca 2+ -ATPase activity. Ca 2+ -ATPase activity was measured at different [Ca 2+ ] in the presence of the following: 100 n m calmodulin (▴) as a positive control for PMCA activation; 5 μ

Techniques Used: Activity Assay, Positive Control, Activation Assay

Time course of actin polymerization in Ca 2+ -ATPase and E P experiments. Actin polymerization (6% pyrene-label) was measured as a function of G-actin concentration as follows: ●, 5.00; ○, 2.50; ▾, 1.25; and Δ, 0.62 μ
Figure Legend Snippet: Time course of actin polymerization in Ca 2+ -ATPase and E P experiments. Actin polymerization (6% pyrene-label) was measured as a function of G-actin concentration as follows: ●, 5.00; ○, 2.50; ▾, 1.25; and Δ, 0.62 μ

Techniques Used: Concentration Assay

Related Articles

Electron Microscopy:

Article Title: Characterization of Three Full-length Human Nonmuscle Myosin II Paralogs *
Article Snippet: .. Keywords: Actin, ATPases, Cytoskeleton, Electron Microscopy (EM), Molecular Motors, Myosin ..

Article Title: The Bacterial Actin MamK
Article Snippet: .. Keywords: Actin, ATPases, Cytoskeleton, Electron Microscopy (EM), Microbiology, Bacterial Actin, Magnetosomes, Magnetotactic Bacteria ..

Functional Assay:

Article Title: Proteomic Profiling Identifies the SIM-associated Complex of KSHV-encoded LANA
Article Snippet: .. Analysis of the functional category of identified proteins revealed that the LANASIM and LANANC polypeptides recruit similar functional activities of proteins including ATPases (LANANC : DD16; LANASIM : ATP2A2, ATP13A1, ATP synthase ATP5A1 and ATP5B), RNA helicases (LANANC : DD16; LANASIM : DHX9, XRCC5, SUV3, DDX17, DDX39A, and DDX6), Chaperon (LANANC : DJC8, Nucleophosmin; LANASIM : Hsp90, Hsp70L), and cytoskeleton (LANANC : Merlin, Moesin, Periplakin, Nebulin; LANASIM : Keratin1, Keratin 2, Keratin 6B, Keratin 9, Keratin 10, Keratin 19)[ ]. ..

other:

Article Title: A Common Substrate Recognition Mode Conserved between Katanin p60 and VPS4 Governs Microtubule Severing and Membrane Skeleton Reorganization *
Article Snippet: Keywords: ATPases, Cytoskeleton, Endosomes, Membrane Trafficking, Microtubules

Article Title: Ion-dependent Polymerization Differences between Mammalian ?- and ?-Nonmuscle Actin Isoforms *
Article Snippet: Keywords: Actin, ATPases, Calcium, Cytoskeleton, Microfilaments, Ion Dependence, Isoactins, Nonmuscle Cell, Nucleotide Exchange, Polymerization

Article Title: Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction *
Article Snippet: Keywords: Actin, ATPases, Calcium, Calmodulin, Cytoskeleton, Transport

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Cytoskeleton Inc kinesin atpase assays
    A model for Aurora B regulation of central spindle size. In early anaphase, PRC1 promotes the formation of anti-parallel microtubule overlaps at the central spindle. At the same time Aurora B is transported to these overlap structures by Mklp2, establishing a local high concentration of Aurora B. Phosphorylated KIF4A (KIF4P) suppresses plus-end microtubule dynamics, and thereby limits growth of the central spindle. Motor activity of <t>kinesin</t> family proteins depends on its ability to alternate between ATP-bound and ADP-bound forms using its <t>ATPase</t> activity. The two forms allow kinesins to cycle between high affinity and low affinity toward microtubules, resulting in its movement along microtubules over time ( Friel and Howard, 2012 ). The processivity of a motor (number of steps a motor takes upon encounter with a microtubule) is directly dependent on its ATPase activity (V max ) as well as its affinity toward microtubules (K app MT). Phosphorylation of KIF4A by Aurora B increases its ATPase activity by ∼5,000 fold compared with its ATPase activity in the absence of microtubules. However, this increase in ATPase activity is not accompanied by increased affinity toward the microtubules. Alternatively, Aurora B mediated phosphorylation may enhance the processivity of KIF4A. Concentration of Aurora B activity at the central spindle during anaphase would allow increased processivity of KIF4A specifically at the central spindle, allowing the motor to preferentially reach microtubule plus-ends and suppress microtubule dynamics in this region.
    Kinesin Atpase Assays, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kinesin atpase assays/product/Cytoskeleton Inc
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kinesin atpase assays - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    85
    Cytoskeleton Inc kinesin atpase activity
    A model for Aurora B regulation of central spindle size. In early anaphase, PRC1 promotes the formation of anti-parallel microtubule overlaps at the central spindle. At the same time Aurora B is transported to these overlap structures by Mklp2, establishing a local high concentration of Aurora B. Phosphorylated KIF4A (KIF4P) suppresses plus-end microtubule dynamics, and thereby limits growth of the central spindle. Motor activity of <t>kinesin</t> family proteins depends on its ability to alternate between ATP-bound and ADP-bound forms using its <t>ATPase</t> activity. The two forms allow kinesins to cycle between high affinity and low affinity toward microtubules, resulting in its movement along microtubules over time ( Friel and Howard, 2012 ). The processivity of a motor (number of steps a motor takes upon encounter with a microtubule) is directly dependent on its ATPase activity (V max ) as well as its affinity toward microtubules (K app MT). Phosphorylation of KIF4A by Aurora B increases its ATPase activity by ∼5,000 fold compared with its ATPase activity in the absence of microtubules. However, this increase in ATPase activity is not accompanied by increased affinity toward the microtubules. Alternatively, Aurora B mediated phosphorylation may enhance the processivity of KIF4A. Concentration of Aurora B activity at the central spindle during anaphase would allow increased processivity of KIF4A specifically at the central spindle, allowing the motor to preferentially reach microtubule plus-ends and suppress microtubule dynamics in this region.
    Kinesin Atpase Activity, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kinesin atpase activity/product/Cytoskeleton Inc
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    kinesin atpase activity - by Bioz Stars, 2020-05
    85/100 stars
      Buy from Supplier

    92
    Cytoskeleton Inc para type atpases
    McdA Activity. ( A ) Bacterial two-hybrid between McdA and McdB tagged at their N-termini (X-McdA, X-McdB) or C-termini (McdA-X, McdB-X) on X-gal supplemented plate. ( B ) Comparison of the <t>ATPase</t> activities of His-MBP-McdA, F SopA-His, and P1 ParA in the presence (solid line, filled symbols) and absence (dashed line, open symbols) of nsDNA. ( C ) Coelution of ATPase activity with His-MBP-McdA concentration. Purified His-MBP-McdA was further purified over a Superdex 200 column. Protein concentration (bar) and ATPase activity (line) were measured for each fraction indicated. Inset shows an SDS-PAGE gel of the purified fractions. ( D ) Comparison of ATPase-specific activity of P1 ParA and F SopA-His in the presence (solid line) and absence (dashed line) of nsDNA. ( E ) Concentration dependency on the specific activity of His-MBP-McdA in the presence (solid line) and absence (dashed line) of nsDNA. ( F ) ATPase activity assays of McdA-GFP-His with/without DNA and/or McdB-His. ( G ) Comparison of the ATPase activity of His-MBP-McdA in the presence of increasing McdB concentration with (solid line) or without (dashed line) nsDNA present. Error bars represent SD from at least three independent experiments. 10.7554/eLife.39723.011 Source data for panel B. 10.7554/eLife.39723.012 Source data for panel D. 10.7554/eLife.39723.013 Source data for panel F. 10.7554/eLife.39723.014 Source data for panel G.
    Para Type Atpases, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/para type atpases/product/Cytoskeleton Inc
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    para type atpases - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    92
    Cytoskeleton Inc f actin activated atpase measurements isolated porcine β cardiac myosin
    Effects of R58Q mutation on isolated cardiac myosin and RLC. (A) SDS-PAGE of RLC-depleted porcine <t>β-cardiac</t> myosin, and myosin reconstituted with either WT- and/or R58Q-RLC. The endogenous (eRLC) and recombinant RLC (rRLC) are labelled accordingly. (B) F-actin dependent <t>ATPase</t> activity of RLC-exchanged β-cardiac myosins from (A) (for details see Materials and Methods ). Data points fitted to the Michaelis-Menten equation (solid lines). (C) Protein stability of WT- and R58Q-RLCs were assessed by Micro-scale Thermophoresis (MST) against increasing concentrations of Guanidine Hydrochloride (GdnHCl). Data points between 0 and 3 mol/L GdnHCl were fitted to a Hill equation (solid lines). (D) Binding of WT- and R58Q-RLC to N-terminal (C0C2) domains of cMyBP-C assessed by MST. Means ± SEM (n = 4–6 for C0C2; n = 1 for C3C5).
    F Actin Activated Atpase Measurements Isolated Porcine β Cardiac Myosin, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/f actin activated atpase measurements isolated porcine β cardiac myosin/product/Cytoskeleton Inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    f actin activated atpase measurements isolated porcine β cardiac myosin - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

    Image Search Results


    A model for Aurora B regulation of central spindle size. In early anaphase, PRC1 promotes the formation of anti-parallel microtubule overlaps at the central spindle. At the same time Aurora B is transported to these overlap structures by Mklp2, establishing a local high concentration of Aurora B. Phosphorylated KIF4A (KIF4P) suppresses plus-end microtubule dynamics, and thereby limits growth of the central spindle. Motor activity of kinesin family proteins depends on its ability to alternate between ATP-bound and ADP-bound forms using its ATPase activity. The two forms allow kinesins to cycle between high affinity and low affinity toward microtubules, resulting in its movement along microtubules over time ( Friel and Howard, 2012 ). The processivity of a motor (number of steps a motor takes upon encounter with a microtubule) is directly dependent on its ATPase activity (V max ) as well as its affinity toward microtubules (K app MT). Phosphorylation of KIF4A by Aurora B increases its ATPase activity by ∼5,000 fold compared with its ATPase activity in the absence of microtubules. However, this increase in ATPase activity is not accompanied by increased affinity toward the microtubules. Alternatively, Aurora B mediated phosphorylation may enhance the processivity of KIF4A. Concentration of Aurora B activity at the central spindle during anaphase would allow increased processivity of KIF4A specifically at the central spindle, allowing the motor to preferentially reach microtubule plus-ends and suppress microtubule dynamics in this region.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    doi: 10.1083/jcb.201301094

    Figure Lengend Snippet: A model for Aurora B regulation of central spindle size. In early anaphase, PRC1 promotes the formation of anti-parallel microtubule overlaps at the central spindle. At the same time Aurora B is transported to these overlap structures by Mklp2, establishing a local high concentration of Aurora B. Phosphorylated KIF4A (KIF4P) suppresses plus-end microtubule dynamics, and thereby limits growth of the central spindle. Motor activity of kinesin family proteins depends on its ability to alternate between ATP-bound and ADP-bound forms using its ATPase activity. The two forms allow kinesins to cycle between high affinity and low affinity toward microtubules, resulting in its movement along microtubules over time ( Friel and Howard, 2012 ). The processivity of a motor (number of steps a motor takes upon encounter with a microtubule) is directly dependent on its ATPase activity (V max ) as well as its affinity toward microtubules (K app MT). Phosphorylation of KIF4A by Aurora B increases its ATPase activity by ∼5,000 fold compared with its ATPase activity in the absence of microtubules. However, this increase in ATPase activity is not accompanied by increased affinity toward the microtubules. Alternatively, Aurora B mediated phosphorylation may enhance the processivity of KIF4A. Concentration of Aurora B activity at the central spindle during anaphase would allow increased processivity of KIF4A specifically at the central spindle, allowing the motor to preferentially reach microtubule plus-ends and suppress microtubule dynamics in this region.

    Article Snippet: Reagents for microtubule polymerization and kinesin ATPase assays were obtained from Cytoskeleton, Inc. A benchtop microfuge (5417R; Eppendorf) was used for all centrifugations unless otherwise indicated.

    Techniques: Concentration Assay, Activity Assay

    Phosphorylation by Aurora B increases KIF4A kinesin ATPase activity. (A) Microtubule-binding assays were performed as described in Materials and methods using 10 nM of the full-length KIF4A (KIF4 FL ), or (B) 33 nM of the KIF4A motor domain (KIF4 MD ) in the presence or absence of 10 nM PRC1 or Cdk1-phosphorylated PRC1 as indicated. Equivalent aliquots of the microtubule pellet fraction (P) and supernatant fractions (S) were analyzed by Western blotting. (C) Coomassie blue–stained gels of proteins used in the assays are shown. (D) Kinesin ATPase assays were performed using 50 nM KIF4 FL ( n = 5 independent experiments), (E) 75 nM KIF4 MD ( n = 2 independent experiments), or (F) KIF4 FL in the presence or absence of 50 nM PRC1 ( n = 2 independent experiments). For some conditions, KIF4A was phosphorylated using wild-type active Aurora kinase (Aur WT ) or an inactive “kinase-dead” mutant (Aur KD ). (G) Kinesin ATPase assays were performed using 50 nM KIF4 FL or (H) KIF4 S799A/T801A mutant left untreated or phosphorylated using wild-type active Aurora kinase (Aur WT ) in the presence or absence of 10 µM ZM447439 Aurora B inhibitor ( n = 3 and n = 2 independent experiments, respectively). (I) Kinesin ATPases assays were performed using 50 nM full-length KIF4A or Aurora-phosphorylated KIF4A at different microtubule concentrations in 150 µl final volume for n = 2 independent experiments. Initial ATPase rates were plotted as a function of microtubule concentration. Assuming ATPase activity followed Michael-Menten kinetics, K app and V max were obtained.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    doi: 10.1083/jcb.201301094

    Figure Lengend Snippet: Phosphorylation by Aurora B increases KIF4A kinesin ATPase activity. (A) Microtubule-binding assays were performed as described in Materials and methods using 10 nM of the full-length KIF4A (KIF4 FL ), or (B) 33 nM of the KIF4A motor domain (KIF4 MD ) in the presence or absence of 10 nM PRC1 or Cdk1-phosphorylated PRC1 as indicated. Equivalent aliquots of the microtubule pellet fraction (P) and supernatant fractions (S) were analyzed by Western blotting. (C) Coomassie blue–stained gels of proteins used in the assays are shown. (D) Kinesin ATPase assays were performed using 50 nM KIF4 FL ( n = 5 independent experiments), (E) 75 nM KIF4 MD ( n = 2 independent experiments), or (F) KIF4 FL in the presence or absence of 50 nM PRC1 ( n = 2 independent experiments). For some conditions, KIF4A was phosphorylated using wild-type active Aurora kinase (Aur WT ) or an inactive “kinase-dead” mutant (Aur KD ). (G) Kinesin ATPase assays were performed using 50 nM KIF4 FL or (H) KIF4 S799A/T801A mutant left untreated or phosphorylated using wild-type active Aurora kinase (Aur WT ) in the presence or absence of 10 µM ZM447439 Aurora B inhibitor ( n = 3 and n = 2 independent experiments, respectively). (I) Kinesin ATPases assays were performed using 50 nM full-length KIF4A or Aurora-phosphorylated KIF4A at different microtubule concentrations in 150 µl final volume for n = 2 independent experiments. Initial ATPase rates were plotted as a function of microtubule concentration. Assuming ATPase activity followed Michael-Menten kinetics, K app and V max were obtained.

    Article Snippet: Reagents for microtubule polymerization and kinesin ATPase assays were obtained from Cytoskeleton, Inc. A benchtop microfuge (5417R; Eppendorf) was used for all centrifugations unless otherwise indicated.

    Techniques: Activity Assay, Binding Assay, Western Blot, Staining, Mutagenesis, Concentration Assay

    A model for Aurora B regulation of central spindle size. In early anaphase, PRC1 promotes the formation of anti-parallel microtubule overlaps at the central spindle. At the same time Aurora B is transported to these overlap structures by Mklp2, establishing a local high concentration of Aurora B. Phosphorylated KIF4A (KIF4P) suppresses plus-end microtubule dynamics, and thereby limits growth of the central spindle. Motor activity of kinesin family proteins depends on its ability to alternate between ATP-bound and ADP-bound forms using its ATPase activity. The two forms allow kinesins to cycle between high affinity and low affinity toward microtubules, resulting in its movement along microtubules over time ( Friel and Howard, 2012 ). The processivity of a motor (number of steps a motor takes upon encounter with a microtubule) is directly dependent on its ATPase activity (V max ) as well as its affinity toward microtubules (K app MT). Phosphorylation of KIF4A by Aurora B increases its ATPase activity by ∼5,000 fold compared with its ATPase activity in the absence of microtubules. However, this increase in ATPase activity is not accompanied by increased affinity toward the microtubules. Alternatively, Aurora B mediated phosphorylation may enhance the processivity of KIF4A. Concentration of Aurora B activity at the central spindle during anaphase would allow increased processivity of KIF4A specifically at the central spindle, allowing the motor to preferentially reach microtubule plus-ends and suppress microtubule dynamics in this region.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    doi: 10.1083/jcb.201301094

    Figure Lengend Snippet: A model for Aurora B regulation of central spindle size. In early anaphase, PRC1 promotes the formation of anti-parallel microtubule overlaps at the central spindle. At the same time Aurora B is transported to these overlap structures by Mklp2, establishing a local high concentration of Aurora B. Phosphorylated KIF4A (KIF4P) suppresses plus-end microtubule dynamics, and thereby limits growth of the central spindle. Motor activity of kinesin family proteins depends on its ability to alternate between ATP-bound and ADP-bound forms using its ATPase activity. The two forms allow kinesins to cycle between high affinity and low affinity toward microtubules, resulting in its movement along microtubules over time ( Friel and Howard, 2012 ). The processivity of a motor (number of steps a motor takes upon encounter with a microtubule) is directly dependent on its ATPase activity (V max ) as well as its affinity toward microtubules (K app MT). Phosphorylation of KIF4A by Aurora B increases its ATPase activity by ∼5,000 fold compared with its ATPase activity in the absence of microtubules. However, this increase in ATPase activity is not accompanied by increased affinity toward the microtubules. Alternatively, Aurora B mediated phosphorylation may enhance the processivity of KIF4A. Concentration of Aurora B activity at the central spindle during anaphase would allow increased processivity of KIF4A specifically at the central spindle, allowing the motor to preferentially reach microtubule plus-ends and suppress microtubule dynamics in this region.

    Article Snippet: Kinesin motor ATPase assays A commercial enzyme–linked inorganic phosphate assay was used to measure kinesin ATPase activity (Cytoskeleton, Inc.).

    Techniques: Concentration Assay, Activity Assay

    Phosphorylation by Aurora B increases KIF4A kinesin ATPase activity. (A) Microtubule-binding assays were performed as described in Materials and methods using 10 nM of the full-length KIF4A (KIF4 FL ), or (B) 33 nM of the KIF4A motor domain (KIF4 MD ) in the presence or absence of 10 nM PRC1 or Cdk1-phosphorylated PRC1 as indicated. Equivalent aliquots of the microtubule pellet fraction (P) and supernatant fractions (S) were analyzed by Western blotting. (C) Coomassie blue–stained gels of proteins used in the assays are shown. (D) Kinesin ATPase assays were performed using 50 nM KIF4 FL ( n = 5 independent experiments), (E) 75 nM KIF4 MD ( n = 2 independent experiments), or (F) KIF4 FL in the presence or absence of 50 nM PRC1 ( n = 2 independent experiments). For some conditions, KIF4A was phosphorylated using wild-type active Aurora kinase (Aur WT ) or an inactive “kinase-dead” mutant (Aur KD ). (G) Kinesin ATPase assays were performed using 50 nM KIF4 FL or (H) KIF4 S799A/T801A mutant left untreated or phosphorylated using wild-type active Aurora kinase (Aur WT ) in the presence or absence of 10 µM ZM447439 Aurora B inhibitor ( n = 3 and n = 2 independent experiments, respectively). (I) Kinesin ATPases assays were performed using 50 nM full-length KIF4A or Aurora-phosphorylated KIF4A at different microtubule concentrations in 150 µl final volume for n = 2 independent experiments. Initial ATPase rates were plotted as a function of microtubule concentration. Assuming ATPase activity followed Michael-Menten kinetics, K app and V max were obtained.

    Journal: The Journal of Cell Biology

    Article Title: Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

    doi: 10.1083/jcb.201301094

    Figure Lengend Snippet: Phosphorylation by Aurora B increases KIF4A kinesin ATPase activity. (A) Microtubule-binding assays were performed as described in Materials and methods using 10 nM of the full-length KIF4A (KIF4 FL ), or (B) 33 nM of the KIF4A motor domain (KIF4 MD ) in the presence or absence of 10 nM PRC1 or Cdk1-phosphorylated PRC1 as indicated. Equivalent aliquots of the microtubule pellet fraction (P) and supernatant fractions (S) were analyzed by Western blotting. (C) Coomassie blue–stained gels of proteins used in the assays are shown. (D) Kinesin ATPase assays were performed using 50 nM KIF4 FL ( n = 5 independent experiments), (E) 75 nM KIF4 MD ( n = 2 independent experiments), or (F) KIF4 FL in the presence or absence of 50 nM PRC1 ( n = 2 independent experiments). For some conditions, KIF4A was phosphorylated using wild-type active Aurora kinase (Aur WT ) or an inactive “kinase-dead” mutant (Aur KD ). (G) Kinesin ATPase assays were performed using 50 nM KIF4 FL or (H) KIF4 S799A/T801A mutant left untreated or phosphorylated using wild-type active Aurora kinase (Aur WT ) in the presence or absence of 10 µM ZM447439 Aurora B inhibitor ( n = 3 and n = 2 independent experiments, respectively). (I) Kinesin ATPases assays were performed using 50 nM full-length KIF4A or Aurora-phosphorylated KIF4A at different microtubule concentrations in 150 µl final volume for n = 2 independent experiments. Initial ATPase rates were plotted as a function of microtubule concentration. Assuming ATPase activity followed Michael-Menten kinetics, K app and V max were obtained.

    Article Snippet: Kinesin motor ATPase assays A commercial enzyme–linked inorganic phosphate assay was used to measure kinesin ATPase activity (Cytoskeleton, Inc.).

    Techniques: Activity Assay, Binding Assay, Western Blot, Staining, Mutagenesis, Concentration Assay

    McdA Activity. ( A ) Bacterial two-hybrid between McdA and McdB tagged at their N-termini (X-McdA, X-McdB) or C-termini (McdA-X, McdB-X) on X-gal supplemented plate. ( B ) Comparison of the ATPase activities of His-MBP-McdA, F SopA-His, and P1 ParA in the presence (solid line, filled symbols) and absence (dashed line, open symbols) of nsDNA. ( C ) Coelution of ATPase activity with His-MBP-McdA concentration. Purified His-MBP-McdA was further purified over a Superdex 200 column. Protein concentration (bar) and ATPase activity (line) were measured for each fraction indicated. Inset shows an SDS-PAGE gel of the purified fractions. ( D ) Comparison of ATPase-specific activity of P1 ParA and F SopA-His in the presence (solid line) and absence (dashed line) of nsDNA. ( E ) Concentration dependency on the specific activity of His-MBP-McdA in the presence (solid line) and absence (dashed line) of nsDNA. ( F ) ATPase activity assays of McdA-GFP-His with/without DNA and/or McdB-His. ( G ) Comparison of the ATPase activity of His-MBP-McdA in the presence of increasing McdB concentration with (solid line) or without (dashed line) nsDNA present. Error bars represent SD from at least three independent experiments. 10.7554/eLife.39723.011 Source data for panel B. 10.7554/eLife.39723.012 Source data for panel D. 10.7554/eLife.39723.013 Source data for panel F. 10.7554/eLife.39723.014 Source data for panel G.

    Journal: eLife

    Article Title: Protein gradients on the nucleoid position the carbon-fixing organelles of cyanobacteria

    doi: 10.7554/eLife.39723

    Figure Lengend Snippet: McdA Activity. ( A ) Bacterial two-hybrid between McdA and McdB tagged at their N-termini (X-McdA, X-McdB) or C-termini (McdA-X, McdB-X) on X-gal supplemented plate. ( B ) Comparison of the ATPase activities of His-MBP-McdA, F SopA-His, and P1 ParA in the presence (solid line, filled symbols) and absence (dashed line, open symbols) of nsDNA. ( C ) Coelution of ATPase activity with His-MBP-McdA concentration. Purified His-MBP-McdA was further purified over a Superdex 200 column. Protein concentration (bar) and ATPase activity (line) were measured for each fraction indicated. Inset shows an SDS-PAGE gel of the purified fractions. ( D ) Comparison of ATPase-specific activity of P1 ParA and F SopA-His in the presence (solid line) and absence (dashed line) of nsDNA. ( E ) Concentration dependency on the specific activity of His-MBP-McdA in the presence (solid line) and absence (dashed line) of nsDNA. ( F ) ATPase activity assays of McdA-GFP-His with/without DNA and/or McdB-His. ( G ) Comparison of the ATPase activity of His-MBP-McdA in the presence of increasing McdB concentration with (solid line) or without (dashed line) nsDNA present. Error bars represent SD from at least three independent experiments. 10.7554/eLife.39723.011 Source data for panel B. 10.7554/eLife.39723.012 Source data for panel D. 10.7554/eLife.39723.013 Source data for panel F. 10.7554/eLife.39723.014 Source data for panel G.

    Article Snippet: Largely influenced by models for DNA segregation by ParA-type ATPases at the time, the observation that C-terminally tagged McdA (McdA-GFP) oscillated in vivo and that carboxysomes were mispositioned following the disruption of McdA or MreB (an actin-related component of the cytoskeleton) led to a widely-adopted hypothesis that carboxysomes were positioned by a cytoskeletal mechanism ( ; ; ; ).

    Techniques: Activity Assay, Concentration Assay, Purification, Protein Concentration, SDS Page

    Effects of R58Q mutation on isolated cardiac myosin and RLC. (A) SDS-PAGE of RLC-depleted porcine β-cardiac myosin, and myosin reconstituted with either WT- and/or R58Q-RLC. The endogenous (eRLC) and recombinant RLC (rRLC) are labelled accordingly. (B) F-actin dependent ATPase activity of RLC-exchanged β-cardiac myosins from (A) (for details see Materials and Methods ). Data points fitted to the Michaelis-Menten equation (solid lines). (C) Protein stability of WT- and R58Q-RLCs were assessed by Micro-scale Thermophoresis (MST) against increasing concentrations of Guanidine Hydrochloride (GdnHCl). Data points between 0 and 3 mol/L GdnHCl were fitted to a Hill equation (solid lines). (D) Binding of WT- and R58Q-RLC to N-terminal (C0C2) domains of cMyBP-C assessed by MST. Means ± SEM (n = 4–6 for C0C2; n = 1 for C3C5).

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle

    doi: 10.1016/j.yjmcc.2018.02.009

    Figure Lengend Snippet: Effects of R58Q mutation on isolated cardiac myosin and RLC. (A) SDS-PAGE of RLC-depleted porcine β-cardiac myosin, and myosin reconstituted with either WT- and/or R58Q-RLC. The endogenous (eRLC) and recombinant RLC (rRLC) are labelled accordingly. (B) F-actin dependent ATPase activity of RLC-exchanged β-cardiac myosins from (A) (for details see Materials and Methods ). Data points fitted to the Michaelis-Menten equation (solid lines). (C) Protein stability of WT- and R58Q-RLCs were assessed by Micro-scale Thermophoresis (MST) against increasing concentrations of Guanidine Hydrochloride (GdnHCl). Data points between 0 and 3 mol/L GdnHCl were fitted to a Hill equation (solid lines). (D) Binding of WT- and R58Q-RLC to N-terminal (C0C2) domains of cMyBP-C assessed by MST. Means ± SEM (n = 4–6 for C0C2; n = 1 for C3C5).

    Article Snippet: 2.6 Reconstitution of recombinant RLCs into isolated β-cardiac myosin, and F-actin activated ATPase measurements Isolated porcine β-cardiac myosin (Cytoskeleton Inc., Denver, CO 80223, USA; MYO3-A) was depleted of its endogenous RLCs and reconstituted with recombinant RLCs as described previously [ ].

    Techniques: Mutagenesis, Isolation, SDS Page, Recombinant, Activity Assay, Microscale Thermophoresis, Binding Assay