β glycerophosphate  (Roche)


Bioz Verified Symbol Roche is a verified supplier
Bioz Manufacturer Symbol Roche manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93

    Structured Review

    Roche β glycerophosphate
    β Glycerophosphate, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β glycerophosphate/product/Roche
    Average 93 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    β glycerophosphate - by Bioz Stars, 2020-09
    93/100 stars

    Images

    Related Articles

    Lysis:

    Article Title: Skeletal muscle-specific Prmt1 deletion causes muscle atrophy via deregulation of the PRMT6-FOXO3 axis
    Article Snippet: .. The cells were lysed in lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma-Aldrich, T8787), 2.5 mM Sodium pyrophosphate, 50 mM NaF, 5 mM β-glycerophosphate, 1 mM Na3 VO4 , proteinase inhibitor cocktail (Roche, 04693132001). .. The lysates were incubated with anti-flag M2 affinity gel (Sigma-aldrich, A2220) at 4°C.

    Article Title: Impaired Priming and Activation of the Neutrophil NADPH Oxidase in Patients with IRAK4- or NEMO-deficiency *
    Article Snippet: .. The treated cells were centrifuged at 1000 × g for 10 min at 4°C and the pellets were lysed in Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA) containing 20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1mM EGTA, 1 mM Na2 EDTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 1 μg/ml leupeptin, 1 mM β-glycerophosphate and Complete Protease Inhibitor Cocktail (Roche Diagnostics Corp., Indianapolis, IN). .. Immunoprecipitation was performed by incubating cleared supernatant with 5 μg mouse anti-p47phox antibody for 16 hrs at 4°C with constant rotation.

    Article Title: Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy, et al. Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy
    Article Snippet: .. Briefly, cultured cell pellets were lysed with lysis buffer (20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X‐100, 10% glycerol, 1 mmol/L EDTA, 50 mmol/L NaF, 50 mmol/L β‐glycerophosphate, 1 mmol/L Na3 VO4 , 1× cOmplete Protease Inhibitor Cocktail; Roche). ..

    Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling
    Article Snippet: .. Cells were lysed in lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40 [v/v], 0.5% sodium deoxycholate [w/v], 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM NaF, 2 mM Na3 VO4 , and Roche Complete Protease Inhibitor Cocktail Tablets). ..

    Cell Culture:

    Article Title: Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy, et al. Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy
    Article Snippet: .. Briefly, cultured cell pellets were lysed with lysis buffer (20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X‐100, 10% glycerol, 1 mmol/L EDTA, 50 mmol/L NaF, 50 mmol/L β‐glycerophosphate, 1 mmol/L Na3 VO4 , 1× cOmplete Protease Inhibitor Cocktail; Roche). ..

    Protease Inhibitor:

    Article Title: Leptin controls adipose tissue lipogenesis via central, STAT3-independent mechanisms
    Article Snippet: .. We homogenized WAT in 20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 mM sodium fluoride, 40 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 2 mM orthovanadate, 0.5% NP-40 and complete protease inhibitor cocktail (Roche) and centrifuged at 12,000 g for 15 min, and we harvested the supernatant while carefully avoiding the lipid layer on top. .. We measured protein concentration with a BCA protein quantification kit (Pierce).

    Article Title: Impaired Priming and Activation of the Neutrophil NADPH Oxidase in Patients with IRAK4- or NEMO-deficiency *
    Article Snippet: .. The treated cells were centrifuged at 1000 × g for 10 min at 4°C and the pellets were lysed in Cell Lysis Buffer (Cell Signaling Technology, Inc., Danvers, MA) containing 20 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1mM EGTA, 1 mM Na2 EDTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 1 μg/ml leupeptin, 1 mM β-glycerophosphate and Complete Protease Inhibitor Cocktail (Roche Diagnostics Corp., Indianapolis, IN). .. Immunoprecipitation was performed by incubating cleared supernatant with 5 μg mouse anti-p47phox antibody for 16 hrs at 4°C with constant rotation.

    Article Title: Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy, et al. Generation and evaluation of a chimeric antibody against coxsackievirus and adenovirus receptor for cancer therapy
    Article Snippet: .. Briefly, cultured cell pellets were lysed with lysis buffer (20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X‐100, 10% glycerol, 1 mmol/L EDTA, 50 mmol/L NaF, 50 mmol/L β‐glycerophosphate, 1 mmol/L Na3 VO4 , 1× cOmplete Protease Inhibitor Cocktail; Roche). ..

    Article Title: Functional diversification of Ser-Arg rich protein kinases to control ubiquitin-dependent neurodevelopmental signalling
    Article Snippet: .. Cells were lysed in lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40 [v/v], 0.5% sodium deoxycholate [w/v], 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM NaF, 2 mM Na3 VO4 , and Roche Complete Protease Inhibitor Cocktail Tablets). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Roche beta glycerophosphate
    Pharmacological inhibition of Slc10a2 induces altered activity of important signal transduction pathways in ob/ob liver. The activation state of selected kinases important in glucose and lipid metabolism were investigated in individual liver protein extracts by phosphorylation site-specific antibodies in Slc10a2 inhibitor-treated and control ob/ob mice. ( A ) Western blot against liver pAkt (ser 473), the same membrane was stripped and reprobed with an antibody against total amount of Akt, ( B ) western blot against liver pMek1/2 (ser 217/221) and pErk1/2 (Thr 202/Tyr 204), the same membranes were consecutively stripped and reprobed with antibodies against total Mek1/2 and Erk1/2. All membranes were finally reprobed with an antibody against <t>beta-actin.</t> Blots are representative of four individual runs.
    Beta Glycerophosphate, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta glycerophosphate/product/Roche
    Average 92 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    beta glycerophosphate - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    93
    Roche np40 buffer
    Detection of histones in peripheral T lymphocytes by western blot. ( A ) Purified naive T cells were left untreated or stimulated with anti-CD3 antibodies for 1, 3, 6, 12 or 24 h. Half of each sample was either acid extracted overnight or lysed using <t>NP40</t>
    Np40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np40 buffer/product/Roche
    Average 93 stars, based on 101 article reviews
    Price from $9.99 to $1999.99
    np40 buffer - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    85
    Roche cdc15 cdk1 kinase buffer
    Model for the function of <t>Cdk1</t> at the mSPB. In early anaphase Cdc14 becomes released from the nucleolus by the FEAR pathway ( Stegmeier et al., 2002 ). <t>Cdc15</t> and Mob1 become partially dephosphorylated (dashed lines). In addition, Tem1 binds to the mSPB ( Molk et al., 2004 ) and recruits nonphosphorylated Cdc15 to this SPB. Cdc15 directs Cdk1 and Dbf2–Mob1 kinases to the mSPB. We propose that the close vicinity of the proteins at the mSPB leads to phosphorylation of Mob1 and Cdc15 by Cdk1. Cdk1-phosphorylated Cdc15 dissociates from the mSPB restricting Cdk1 at the mSPB (symbolized as inhibition of Cdc15). Phosphorylation of Mob1 by Cdk1 leads to a decrease in Dbf2–Mob1 kinase activity. These events restrict full activation of the MEN at the mSPB. At the dSPB, the Bfa1–Bub2 complex inhibits activation of Tem1 ( Bardin et al., 2000 ; Pereira et al., 2000 ). In late anaphase the increase in Cdc14 activity and the decrease in Cdk1 activity disrupt the regulation loop between Cdk1 and Cdc15 at the mSPB (thick dashed lines symbolize complete dephosphorylation by Cdc14). This together with the phosphorylation of Bfa1 by Cdc5 polo-like kinase at the dSPB ( Hu et al., 2001 ; Geymonat et al., 2003 ; Maekawa et al., 2007 ) allows full activation of the MEN. Dbf2–Mob1 kinase then phosphorylates Cdc14 ( Mohl et al., 2009 ).
    Cdc15 Cdk1 Kinase Buffer, supplied by Roche, 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/cdc15 cdk1 kinase buffer/product/Roche
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cdc15 cdk1 kinase buffer - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results


    Pharmacological inhibition of Slc10a2 induces altered activity of important signal transduction pathways in ob/ob liver. The activation state of selected kinases important in glucose and lipid metabolism were investigated in individual liver protein extracts by phosphorylation site-specific antibodies in Slc10a2 inhibitor-treated and control ob/ob mice. ( A ) Western blot against liver pAkt (ser 473), the same membrane was stripped and reprobed with an antibody against total amount of Akt, ( B ) western blot against liver pMek1/2 (ser 217/221) and pErk1/2 (Thr 202/Tyr 204), the same membranes were consecutively stripped and reprobed with antibodies against total Mek1/2 and Erk1/2. All membranes were finally reprobed with an antibody against beta-actin. Blots are representative of four individual runs.

    Journal: PLoS ONE

    Article Title: Inhibition of Intestinal Bile Acid Transporter Slc10a2 Improves Triglyceride Metabolism and Normalizes Elevated Plasma Glucose Levels in Mice

    doi: 10.1371/journal.pone.0037787

    Figure Lengend Snippet: Pharmacological inhibition of Slc10a2 induces altered activity of important signal transduction pathways in ob/ob liver. The activation state of selected kinases important in glucose and lipid metabolism were investigated in individual liver protein extracts by phosphorylation site-specific antibodies in Slc10a2 inhibitor-treated and control ob/ob mice. ( A ) Western blot against liver pAkt (ser 473), the same membrane was stripped and reprobed with an antibody against total amount of Akt, ( B ) western blot against liver pMek1/2 (ser 217/221) and pErk1/2 (Thr 202/Tyr 204), the same membranes were consecutively stripped and reprobed with antibodies against total Mek1/2 and Erk1/2. All membranes were finally reprobed with an antibody against beta-actin. Blots are representative of four individual runs.

    Article Snippet: To analyze phosphorylated liver proteins by Western blot, total liver protein homogenates were prepared from frozen tissue by homogenization using a polytron followed by sonication in a buffer containing 20 mM Tris-Hcl, pH 7,4, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 2 mM EDTA, 25 mM beta-glycerophosphate, 20 mM sodium floride, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 1 mM benzamidine, 1 mM phenyl-methylsulfonyl fluoride, 0.5 mM leupeptin, Complete protease inhibitor (Roche), and then centrifuged at 14 000 rpm in a microcentrifuge, and the supernatant was recovered.

    Techniques: Inhibition, Activity Assay, Transduction, Activation Assay, Mouse Assay, Western Blot

    Signaling pathways showing the genes/groups of genes up-regulated (pink) in BCP group compared to oil group. (a) Sonic hedgehog signaling (shh) pathway, (b) planar cell polarity (PCP) signaling pathway, (c) fibroblast growth factor (FGF) signaling pathway, and (d) Wnt beta-catenin signaling pathway.

    Journal: bioRxiv

    Article Title: Beta-caryophyllene enhances wound healing through multiple routes

    doi: 10.1101/611046

    Figure Lengend Snippet: Signaling pathways showing the genes/groups of genes up-regulated (pink) in BCP group compared to oil group. (a) Sonic hedgehog signaling (shh) pathway, (b) planar cell polarity (PCP) signaling pathway, (c) fibroblast growth factor (FGF) signaling pathway, and (d) Wnt beta-catenin signaling pathway.

    Article Snippet: The dissected OE was transferred to 1.35 mL of homogenization buffer (150 mM KCl, 5 mM MgCl2 , 10 mM HEPES [pH 7.4], 100 nM Calyculin A, 2 mM DTT, 100 U/mL RNasin (Promega), 100 μg/mL cycloheximide, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, protease inhibitor (Roche, 1 tablet per 10 ml)) and homogenized three times at 250 rpm and nine times at 750 rpm (Glas-Col).

    Techniques:

    Beta-caryophyllene standard (Sigma-Aldrich) composition/GC-MS. 1: cubebene, 2, 4, 5, 7, 8: sesquiterpenes of MW 204, 3: copaene, 6: BCP, 9: neoclovene, 10: α-caryophyllene, 11: 9-epi(E)-caryophyllene, 12: caryophyllene oxide. See S2 Table for details.

    Journal: bioRxiv

    Article Title: Beta-caryophyllene enhances wound healing through multiple routes

    doi: 10.1101/611046

    Figure Lengend Snippet: Beta-caryophyllene standard (Sigma-Aldrich) composition/GC-MS. 1: cubebene, 2, 4, 5, 7, 8: sesquiterpenes of MW 204, 3: copaene, 6: BCP, 9: neoclovene, 10: α-caryophyllene, 11: 9-epi(E)-caryophyllene, 12: caryophyllene oxide. See S2 Table for details.

    Article Snippet: The dissected OE was transferred to 1.35 mL of homogenization buffer (150 mM KCl, 5 mM MgCl2 , 10 mM HEPES [pH 7.4], 100 nM Calyculin A, 2 mM DTT, 100 U/mL RNasin (Promega), 100 μg/mL cycloheximide, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, protease inhibitor (Roche, 1 tablet per 10 ml)) and homogenized three times at 250 rpm and nine times at 750 rpm (Glas-Col).

    Techniques: Gas Chromatography-Mass Spectrometry

    Detection of histones in peripheral T lymphocytes by western blot. ( A ) Purified naive T cells were left untreated or stimulated with anti-CD3 antibodies for 1, 3, 6, 12 or 24 h. Half of each sample was either acid extracted overnight or lysed using NP40

    Journal: The EMBO Journal

    Article Title: Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence

    doi: 10.1038/emboj.2010.314

    Figure Lengend Snippet: Detection of histones in peripheral T lymphocytes by western blot. ( A ) Purified naive T cells were left untreated or stimulated with anti-CD3 antibodies for 1, 3, 6, 12 or 24 h. Half of each sample was either acid extracted overnight or lysed using NP40

    Article Snippet: Whole-cell extracts were prepared by incubating cells in NP40 buffer (1% NP40, 50 mM Tris, pH 7.5, 100 mM NaCl, 50 mM NaF, 40 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM EDTA) supplemented with Complete Mini protease inhibitors (Roche) for 30 min at 4°C.

    Techniques: Western Blot, Purification

    Model for the function of Cdk1 at the mSPB. In early anaphase Cdc14 becomes released from the nucleolus by the FEAR pathway ( Stegmeier et al., 2002 ). Cdc15 and Mob1 become partially dephosphorylated (dashed lines). In addition, Tem1 binds to the mSPB ( Molk et al., 2004 ) and recruits nonphosphorylated Cdc15 to this SPB. Cdc15 directs Cdk1 and Dbf2–Mob1 kinases to the mSPB. We propose that the close vicinity of the proteins at the mSPB leads to phosphorylation of Mob1 and Cdc15 by Cdk1. Cdk1-phosphorylated Cdc15 dissociates from the mSPB restricting Cdk1 at the mSPB (symbolized as inhibition of Cdc15). Phosphorylation of Mob1 by Cdk1 leads to a decrease in Dbf2–Mob1 kinase activity. These events restrict full activation of the MEN at the mSPB. At the dSPB, the Bfa1–Bub2 complex inhibits activation of Tem1 ( Bardin et al., 2000 ; Pereira et al., 2000 ). In late anaphase the increase in Cdc14 activity and the decrease in Cdk1 activity disrupt the regulation loop between Cdk1 and Cdc15 at the mSPB (thick dashed lines symbolize complete dephosphorylation by Cdc14). This together with the phosphorylation of Bfa1 by Cdc5 polo-like kinase at the dSPB ( Hu et al., 2001 ; Geymonat et al., 2003 ; Maekawa et al., 2007 ) allows full activation of the MEN. Dbf2–Mob1 kinase then phosphorylates Cdc14 ( Mohl et al., 2009 ).

    Journal: The Journal of Cell Biology

    Article Title: Mutual regulation of cyclin-dependent kinase and the mitotic exit network

    doi: 10.1083/jcb.200911128

    Figure Lengend Snippet: Model for the function of Cdk1 at the mSPB. In early anaphase Cdc14 becomes released from the nucleolus by the FEAR pathway ( Stegmeier et al., 2002 ). Cdc15 and Mob1 become partially dephosphorylated (dashed lines). In addition, Tem1 binds to the mSPB ( Molk et al., 2004 ) and recruits nonphosphorylated Cdc15 to this SPB. Cdc15 directs Cdk1 and Dbf2–Mob1 kinases to the mSPB. We propose that the close vicinity of the proteins at the mSPB leads to phosphorylation of Mob1 and Cdc15 by Cdk1. Cdk1-phosphorylated Cdc15 dissociates from the mSPB restricting Cdk1 at the mSPB (symbolized as inhibition of Cdc15). Phosphorylation of Mob1 by Cdk1 leads to a decrease in Dbf2–Mob1 kinase activity. These events restrict full activation of the MEN at the mSPB. At the dSPB, the Bfa1–Bub2 complex inhibits activation of Tem1 ( Bardin et al., 2000 ; Pereira et al., 2000 ). In late anaphase the increase in Cdc14 activity and the decrease in Cdk1 activity disrupt the regulation loop between Cdk1 and Cdc15 at the mSPB (thick dashed lines symbolize complete dephosphorylation by Cdc14). This together with the phosphorylation of Bfa1 by Cdc5 polo-like kinase at the dSPB ( Hu et al., 2001 ; Geymonat et al., 2003 ; Maekawa et al., 2007 ) allows full activation of the MEN. Dbf2–Mob1 kinase then phosphorylates Cdc14 ( Mohl et al., 2009 ).

    Article Snippet: In , the beads with ∼80 ng Dbf2–Mob1 were incubated in 40 µl Cdc15/Cdk1 kinase buffer (50 mM Hepes-KOH, pH 7.5, 100 mM NaCl, 10 mM MgCl2 , 2.5 mM MnCl2 , 5 mM β-glycerophosphate, and 1 mM DTT) containing 5 mM ATP and complete EDTA-free protease inhibitor cocktail (Roche) for 1 h at 30°C with 3 ng of Cdc15 or Cdk1–Clb2 or without kinase (first reaction).

    Techniques: Inhibition, Activity Assay, Activation Assay, De-Phosphorylation Assay

    SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase ( D’Aquino et al., 2005 ). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15 , n = 43 for cdc15-as1 . The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67 , and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.

    Journal: The Journal of Cell Biology

    Article Title: Mutual regulation of cyclin-dependent kinase and the mitotic exit network

    doi: 10.1083/jcb.200911128

    Figure Lengend Snippet: SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase ( D’Aquino et al., 2005 ). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15 , n = 43 for cdc15-as1 . The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67 , and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.

    Article Snippet: In , the beads with ∼80 ng Dbf2–Mob1 were incubated in 40 µl Cdc15/Cdk1 kinase buffer (50 mM Hepes-KOH, pH 7.5, 100 mM NaCl, 10 mM MgCl2 , 2.5 mM MnCl2 , 5 mM β-glycerophosphate, and 1 mM DTT) containing 5 mM ATP and complete EDTA-free protease inhibitor cocktail (Roche) for 1 h at 30°C with 3 ng of Cdc15 or Cdk1–Clb2 or without kinase (first reaction).

    Techniques: Activity Assay, Blocking Assay

    Cdc15 and Cdk1 show mutual regulation at the mSPB. (A) CDC14 and td-cdc14 cells harboring CDC15-GFP mCherry-TUB1 were examined for Cdc15-GFP localization in anaphase. The arrows highlight Cdc15-GFP at SPBs. Bar, 5 µm. (B and C) Anaphase cells of CDC14 CDC15-GFP, td-cdc14 CDC15-GFP , and td-cdc14 CDC15-7A-GFP were grown in YPAD and analyzed for GFP signal at SPBs. Quantified relative fluorescent intensities are summarized in box-and-whisker plots: boxes span between the 25th and 75th percentile with a line at the median; whiskers extend from the 10th to 90th percentile. P-values were calculated using unpaired t tests and indicate significant differences between * or ** marked bars. (B) n > 50 anaphase cells per strain. (C) n > 50 for CDC15-GFP cells and n = 24 for CDC15-7A-GFP cells. (D) CDC15 and CDC15-7A cells were grown in SC medium. Cells in anaphase were examined for Cdk1-GFP localization to SPBs. Bar, 5 µm. (E) Quantification of Cdk1-GFP signal at the mSPB. Relative fluorescent intensities in box-and-whisker plots as in B and C. n > 50 cells were analyzed per strain. (F) Cdk1-GFP protein levels measured with anti-GFP antibody and actin as loading control.

    Journal: The Journal of Cell Biology

    Article Title: Mutual regulation of cyclin-dependent kinase and the mitotic exit network

    doi: 10.1083/jcb.200911128

    Figure Lengend Snippet: Cdc15 and Cdk1 show mutual regulation at the mSPB. (A) CDC14 and td-cdc14 cells harboring CDC15-GFP mCherry-TUB1 were examined for Cdc15-GFP localization in anaphase. The arrows highlight Cdc15-GFP at SPBs. Bar, 5 µm. (B and C) Anaphase cells of CDC14 CDC15-GFP, td-cdc14 CDC15-GFP , and td-cdc14 CDC15-7A-GFP were grown in YPAD and analyzed for GFP signal at SPBs. Quantified relative fluorescent intensities are summarized in box-and-whisker plots: boxes span between the 25th and 75th percentile with a line at the median; whiskers extend from the 10th to 90th percentile. P-values were calculated using unpaired t tests and indicate significant differences between * or ** marked bars. (B) n > 50 anaphase cells per strain. (C) n > 50 for CDC15-GFP cells and n = 24 for CDC15-7A-GFP cells. (D) CDC15 and CDC15-7A cells were grown in SC medium. Cells in anaphase were examined for Cdk1-GFP localization to SPBs. Bar, 5 µm. (E) Quantification of Cdk1-GFP signal at the mSPB. Relative fluorescent intensities in box-and-whisker plots as in B and C. n > 50 cells were analyzed per strain. (F) Cdk1-GFP protein levels measured with anti-GFP antibody and actin as loading control.

    Article Snippet: In , the beads with ∼80 ng Dbf2–Mob1 were incubated in 40 µl Cdc15/Cdk1 kinase buffer (50 mM Hepes-KOH, pH 7.5, 100 mM NaCl, 10 mM MgCl2 , 2.5 mM MnCl2 , 5 mM β-glycerophosphate, and 1 mM DTT) containing 5 mM ATP and complete EDTA-free protease inhibitor cocktail (Roche) for 1 h at 30°C with 3 ng of Cdc15 or Cdk1–Clb2 or without kinase (first reaction).

    Techniques: Whisker Assay

    Cdk1 regulates kinase activity of Dbf2–Mob1. (A) Log-phase cells with the indicated phenotypes were serially diluted 10-fold and spotted onto YPD plates. Plates were incubated for 2 d at 23 or 37°C. (B) Active Dbf2–Mob1 complex was incubated without substrate (lane 1), GST (lane 2), and GST-C-Cdc14 (lane 3) in the presence of γ-[ 32 P]ATP. Shown is an autoradiography. (C) Cdk1–Clb2 kinase inhibits the activation of Dbf2–Mob1 kinase by Cdc15 in vitro. GST-Mob1 in a complex with Dbf2 was incubated with Cdk1–Clb2 or Cdc15 in the first and second kinase reaction as indicated in the figure. After the second reaction, Mob1-Dbf2 kinase assays with GST-C-Cdc14 as substrate and anti-Mob1 immunoblots were performed. The top graph shows the specific Dbf2–Mob1 kinase activity. Shown is the outcome of one out of two independent experiments. Both results were identical. (D) Gal1- CLB2-ΔDB cells were arrested with α-factor in G1 phase in YPAR and released into a synchronized cell cycle at 30°C in YPAR. After ∼60 min, galactose (2%) was added. Cells in anaphase were used for immunoprecipitation of TAP-Dbf2 followed by kinase assays using GST-C-Cdc14 as substrate. Phosphorylation was determined by autoradiography and normalized to immunoprecipitated TAP-Dbf2 (immunoblot anti-TAP). Dbf2–Mob1 kinase activity is shown as mean ± SD of three experiments with the activity of wild-type cells set to 1.

    Journal: The Journal of Cell Biology

    Article Title: Mutual regulation of cyclin-dependent kinase and the mitotic exit network

    doi: 10.1083/jcb.200911128

    Figure Lengend Snippet: Cdk1 regulates kinase activity of Dbf2–Mob1. (A) Log-phase cells with the indicated phenotypes were serially diluted 10-fold and spotted onto YPD plates. Plates were incubated for 2 d at 23 or 37°C. (B) Active Dbf2–Mob1 complex was incubated without substrate (lane 1), GST (lane 2), and GST-C-Cdc14 (lane 3) in the presence of γ-[ 32 P]ATP. Shown is an autoradiography. (C) Cdk1–Clb2 kinase inhibits the activation of Dbf2–Mob1 kinase by Cdc15 in vitro. GST-Mob1 in a complex with Dbf2 was incubated with Cdk1–Clb2 or Cdc15 in the first and second kinase reaction as indicated in the figure. After the second reaction, Mob1-Dbf2 kinase assays with GST-C-Cdc14 as substrate and anti-Mob1 immunoblots were performed. The top graph shows the specific Dbf2–Mob1 kinase activity. Shown is the outcome of one out of two independent experiments. Both results were identical. (D) Gal1- CLB2-ΔDB cells were arrested with α-factor in G1 phase in YPAR and released into a synchronized cell cycle at 30°C in YPAR. After ∼60 min, galactose (2%) was added. Cells in anaphase were used for immunoprecipitation of TAP-Dbf2 followed by kinase assays using GST-C-Cdc14 as substrate. Phosphorylation was determined by autoradiography and normalized to immunoprecipitated TAP-Dbf2 (immunoblot anti-TAP). Dbf2–Mob1 kinase activity is shown as mean ± SD of three experiments with the activity of wild-type cells set to 1.

    Article Snippet: In , the beads with ∼80 ng Dbf2–Mob1 were incubated in 40 µl Cdc15/Cdk1 kinase buffer (50 mM Hepes-KOH, pH 7.5, 100 mM NaCl, 10 mM MgCl2 , 2.5 mM MnCl2 , 5 mM β-glycerophosphate, and 1 mM DTT) containing 5 mM ATP and complete EDTA-free protease inhibitor cocktail (Roche) for 1 h at 30°C with 3 ng of Cdc15 or Cdk1–Clb2 or without kinase (first reaction).

    Techniques: Activity Assay, Incubation, Autoradiography, Activation Assay, In Vitro, Western Blot, Immunoprecipitation