Journal: Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/jci158498

Figure Lengend Snippet: Figure 4. Consumption of a potassium-rich diet increases PP1 protein abundance and decreases I1 protein abundance in the DCT. (A) Immunoblot analysis of renal PP1, I1, and calcineurin in control mice (WT/WT SPAK), or mice with 1 (CA/–) or 2 (CA/CA) CA-SPAK alleles fed a control or high-potassium diet for 4 days. Each lane is from a separate mouse. (B) Quantitative summaries for A; shown is the average protein abundance of PP1, I1, and calcineurin for mice of each genotype on the control diet (orange) or the high-potassium diet (red). Data are relative to control mice on the control diet. (C) PP1A local- ization in control mice harboring 2 WT SPAK alleles (WT/WT). Representative images show PP1A (red) in DCT1, identified by parvalbumin (PV) labeling (green), from WT mice randomized to the control or high-potassium diet for 4 days. Scale bars: 15 μm. Graph shows quantitative image analysis of total cellular PP1A intensity (n = 4 mice/group, each data point represents the average intensity of >30 cells/mouse). *P < 0.05, by 2-tailed Student’s t test. (D) I1 localization in CA-SPAK mice harboring 2 CA-SPAK alleles (CA/CA). Representative images show I1 (red) in DCT1 from homozygous CA-SPAK (WT) mice randomized to the control or high-potassium diet for 4 days. Scale bars: 20 μm. Parvalbumin labeling (green) identified DCT1 (green arrowheads) from the cortical thick ascending limb (TAL), which also expressed I1. Graph shows quantitative image analysis of total cellular I1 intensity (4 mice/group, each data point represents the average intensity of >30 cells/mouse). Data are the mean ± SEM. *P < 0.05, by 2-tailed Student’s t test.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl2, 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: Quantitative Proteomics, Western Blot, Control, Labeling

Journal: Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/jci158498

Figure Lengend Snippet: Figure 5. Potassium liberates PP1 from I1 by 2 mechanisms in control mice. (A) I1 abundance decreased as mice became hyperkalemic, as shown in the immunoblot of I1 in control mice on the control (gray) or high- potassium (orange) diet or in mice treated with amiloride (Amil) for 2 days (red) or 4 days (green). Quantitative summaries show the average I1 abundance and relative abundance versus plasma potassium. Each dot represents a separate mouse. (B) A high-potassium diet decreased I1 phosphorylation at T35 in WT mice. Immunoblot shows p-I1 (T35). Graph shows the quantitative summary of p-I1 relative abundance. *P < 0.05, by 1-way ANOVA followed by Dunnett’s multiple-comparison test (A and B). Data are the mean ± SEM. n = 6 (A); n = 4–5 (B).

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl2, 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: Control, Western Blot, Clinical Proteomics, Phospho-proteomics, Comparison

Journal: Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/jci158498

Figure Lengend Snippet: Figure 6. Potassium regulates PP1A interaction with and dephosphorylation of NCC. (A) In vitro protein phosphatase (PP) assay. Representative immu- noblots of p-NCC and t-NCC are shown for assays of NCC with PP1A (lanes 1 and 2), PP2A (lanes 5 and 6), and calcineurin A (CALNA) or vehicle (lanes 3, 7, and 11). For these studies, NCC was isolated by IP with an anti-NCC antibody (lanes 1, 2, 3, 5, 6, and 7) and compared with the negative control IgG (lanes 4, 8, and 12). (B) PP1A preferentially interacted with NCC in glutathione-agarose affinity chromatography assays with the GST fusion protein of the NCC terminus GST-NCC-(N), but not the negative control GST alone. Shown are representative immunoblot binding assays with recombinant PP1A, PP2A, and CALNA. Input protein phosphatase (lane 1) is shown relative to GST-bound (lane 2) or GST-NCC-(N) (lane 3). (C) A greater amount of PP1A co-immuno precipitated with NCC when the extracellular K+ concentration was high. Representative immunoblots show NCC, PP1A, and p-NCC (T58) in anti-FLAG immunoprecipitated samples relative to input from FLAG-tagged NCC-expressing MDCKI cells incubated with 3.5 mM (control), 0.5 mM (low), or 8 mM (high) K+ buffers. Low-chloride buffer was used as a positive control to elevate p-NCC (T58) levels. Graphs show semiquantitative assessment of NCC, PP1, and p-NCC (T58) in NCC immunoprecipitated samples (bottom, right) relative to input. Data are from 3 individual experiments with 3 replicates for each condition (n = 9). *P < 0.05, relative to the low-K+ condition, by 1-way ANOVA followed by multiple-comparison test. Data are presented as the mean ± SEM. CALNA, calcineurin.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl2, 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: De-Phosphorylation Assay, In Vitro, Isolation, Negative Control, Affinity Chromatography, Western Blot, Binding Assay, Recombinant, Concentration Assay, Immunoprecipitation, Expressing, Incubation, Control, Positive Control, Comparison

Journal: Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/jci158498

Figure Lengend Snippet: Figure 7. Potassium-dependent activation of PP1 drives rapid dephosphorylation of NCC in the DCT, coincident with dephosphorylation (inactivation) of the PP1-inhibitory subunit I1. (A) p-NCC and t-NCC after incubation in control, 2 mM K+ bath (red), or 6 mM K+ bath containing either vehicle (control, orange), tautomycetin (TMC) (blue), or FK506 (FK) (pink). (B) p-I1 and t-I1 as assessed by immunoblotting in kidney slices after incubation in control, 2 mM K+ bath (red), or 6 mM K+ bath containing either vehicle (control, orange), tautomycetin (TMC) (blue), or FK506 (FK) (pink). Parallel studies were performed on slices from control mice (homozygous for WT SPAK, WT/WT) or heterozygous CA-SPAK mice (CA/–), or homozygous CA-SPAK mice fed the indicated diets. A quantitative summary is shown below the blots. Each lane/dot represents a separate mouse. n > 6. *P < 0.05, by 1-way ANOVA with Tukey’s post hoc test. Data are the mean ± SEM.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl2, 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: Activation Assay, De-Phosphorylation Assay, Incubation, Control, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/JCI158498

Figure Lengend Snippet: ( A ) Immunoblot analysis of renal PP1, I1, and calcineurin in control mice (WT/WT SPAK), or mice with 1 (CA/–) or 2 (CA/CA) CA-SPAK alleles fed a control or high-potassium diet for 4 days. Each lane is from a separate mouse. ( B ) Quantitative summaries for A ; shown is the average protein abundance of PP1, I1, and calcineurin for mice of each genotype on the control diet (orange) or the high-potassium diet (red). Data are relative to control mice on the control diet. ( C ) PP1A localization in control mice harboring 2 WT SPAK alleles (WT/WT). Representative images show PP1A (red) in DCT1, identified by parvalbumin (PV) labeling (green), from WT mice randomized to the control or high-potassium diet for 4 days. Scale bars: 15 μm. Graph shows quantitative image analysis of total cellular PP1A intensity ( n = 4 mice/group, each data point represents the average intensity of >30 cells/mouse). * P < 0.05, by 2-tailed Student’s t test. ( D ) I1 localization in CA-SPAK mice harboring 2 CA-SPAK alleles (CA/CA). Representative images show I1 (red) in DCT1 from homozygous CA-SPAK (WT) mice randomized to the control or high-potassium diet for 4 days. Scale bars: 20 μm. Parvalbumin labeling (green) identified DCT1 (green arrowheads) from the cortical thick ascending limb (TAL), which also expressed I1. Graph shows quantitative image analysis of total cellular I1 intensity (4 mice/group, each data point represents the average intensity of >30 cells/mouse). Data are the mean ± SEM. * P < 0.05, by 2-tailed Student’s t test.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl 2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl 2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl 2 , 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: Western Blot, Control, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/JCI158498

Figure Lengend Snippet: ( A ) In vitro protein phosphatase (PP) assay. Representative immunoblots of p-NCC and t-NCC are shown for assays of NCC with PP1A (lanes 1 and 2), PP2A (lanes 5 and 6), and calcineurin A (CALNA) or vehicle (lanes 3, 7, and 11). For these studies, NCC was isolated by IP with an anti-NCC antibody (lanes 1, 2, 3, 5, 6, and 7) and compared with the negative control IgG (lanes 4, 8, and 12). ( B ) PP1A preferentially interacted with NCC in glutathione-agarose affinity chromatography assays with the GST fusion protein of the NCC terminus GST-NCC-(N), but not the negative control GST alone. Shown are representative immunoblot binding assays with recombinant PP1A, PP2A, and CALNA. Input protein phosphatase (lane 1) is shown relative to GST-bound (lane 2) or GST-NCC-(N) (lane 3). ( C ) A greater amount of PP1A co-immunoprecipitated with NCC when the extracellular K + concentration was high. Representative immunoblots show NCC, PP1A, and p-NCC (T58) in anti-FLAG immunoprecipitated samples relative to input from FLAG-tagged NCC-expressing MDCKI cells incubated with 3.5 mM (control), 0.5 mM (low), or 8 mM (high) K + buffers. Low-chloride buffer was used as a positive control to elevate p-NCC (T58) levels. Graphs show semiquantitative assessment of NCC, PP1, and p-NCC (T58) in NCC immunoprecipitated samples (bottom, right) relative to input. Data are from 3 individual experiments with 3 replicates for each condition ( n = 9). * P < 0.05, relative to the low-K + condition, by 1-way ANOVA followed by multiple-comparison test. Data are presented as the mean ± SEM. CALNA, calcineurin.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl 2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl 2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl 2 , 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: In Vitro, Western Blot, Isolation, Negative Control, Affinity Chromatography, Binding Assay, Recombinant, Immunoprecipitation, Concentration Assay, Expressing, Incubation, Control, Positive Control, Comparison

Journal:

Article Title: Wnt-induced dephosphorylation of Axin releases ?-catenin from the Axin complex

doi:

Figure Lengend Snippet: The Axin protein is phosphorylated. (A) PP2A, but not PP1, dephosphorylates the Axin protein in vitro. Axin protein was immunoprecipitated from cell extracts of C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium. The immune complexes were treated with phosphatase buffer only, PP2A, or PP1 and immunoblotted with anti-Axin antibody. (B) The phosphatase inhibitor okadaic acid inhibits the dephosphorylation of Axin. Cell lysates were prepared from C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium and with or without 400 nm okadaic acid and immunoblotted with anti-Axin antibody. (C) Lithium, an inhibitor of GSK-3β, promotes the dephosphorylation of Axin. Cell lysates were prepared from C57MG cells first treated with or without 25 mm LiCl for 4 hr and treated with control (−) or Wnt3A (+)-conditioned medium for an additional 2 hr.

Article Snippet: For phosphatase treatment, the immune complexes prior to boiling were incubated in phosphatase reaction buffer (lysis buffer with 0.1% β-mercaptoethanol, 1 m m EGTA, 0.5 mg/ml BSA, and protease inhibitors) and incubated with 1 unit of PP1 (Promega) or 1 unit of PP2A (Upstate Biotechnologies, Inc.) for 1 hr at 30°C.

Techniques: In Vitro, Immunoprecipitation, De-Phosphorylation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: The H250Y mutation rescues kinase activity of Aurora B gatekeeper mutants. A, multiple sequence alignment of the Aurora kinases in different species. The gatekeeper residue and the second site suppressor mutation found in Ark1 and Ipl1 as well as the newly identified second site suppressor mutation for human and mouse Aurora B are indicated. B, in vitro kinase assay with the indicated recombinant GST-tagged Aurora B proteins and recombinant histone H3 as substrate. Because one master mix of kinase reaction buffer plus histone H3 was used, each reaction was supplemented with the same amount of substrate. Phosphorylation of histone H3 was detected with an antibody specific for phosphoserine 10. Note that even a small amount of Aurora BH250Y results in massive phosphorylation of histone H3. C, mitotic index of paclitaxel-treated U2OS cells transfected with cDNAs encoding the indicated Aurora B mutants. All three mutants are dominant negative over the endogenous kinase and therefore reduce the mitotic delay induced by paclitaxel. D and E, FLAG-tagged human Aurora B mutants (D) and GFP-tagged mouse Aurora B mutants (E) were immunoprecipitated from mitotic U2OS cells. Kinase reactions were performed in the presence of ATP and with or without NA-PP1 (2 μm). Histone H3 was used as substrate. F, the H250Y mutation rescues kinase activity of Aurora B gatekeeper mutants. The double mutants are inhibited by NA-PP1.

Article Snippet: Immunoprecipitated or recombinant kinases were included into a 25-μl reaction containing kinase buffer (10 m m MgCl 2 , 25 m m HEPES, pH 7.5, 25 m m β-glycerophosphate, 0.5 m m DTT and 0.5 m m vanadate), 0.2 mg/ml histone H3 (Roche Diagnostics), and 100 μ m ATP with or without 2 μ m NA-PP1 (Calbiochem/Merck Chemicals).

Techniques: Mutagenesis, Activity Assay, Sequencing, In Vitro, Kinase Assay, Recombinant, Transfection, Dominant Negative Mutation, Immunoprecipitation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: The analog-sensitive Aurora B mutants are inhibited by PP1 analogs in cells. A, U2OS cells expressing the indicated proteins were released from a thymidine-induced G1/S block into medium containing paclitaxel plus or minus the indicated PP1 analogs. Seventeen hours after release, the mitotic index was determined by propidium iodide/MPM2 monoclonal antibody labeling and FACS analysis. B, similar to A, but now different concentrations of NA-PP1 were added to the cells. For each transfected cell population, the mitotic index without PP1 inhibitor was set to 100%, and the relative reduction in mitotic index in the presence of PP1 inhibitor was determined.

Article Snippet: Immunoprecipitated or recombinant kinases were included into a 25-μl reaction containing kinase buffer (10 m m MgCl 2 , 25 m m HEPES, pH 7.5, 25 m m β-glycerophosphate, 0.5 m m DTT and 0.5 m m vanadate), 0.2 mg/ml histone H3 (Roche Diagnostics), and 100 μ m ATP with or without 2 μ m NA-PP1 (Calbiochem/Merck Chemicals).

Techniques: Expressing, Blocking Assay, Antibody Labeling, Transfection

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: Localization of the analog-sensitive Aurora B mutants. U2OS cells expressing the indicating GFP-tagged proteins were fixed 14 h after release from a thymidine-induced G1/S block into the Eg5 inhibitor S-trityl-l-cysteine (44) with or without NA-PP1 (2 μm). Centromeres were detected with human CREST antiserum and endogenous survivin with anti-survivin polyclonal antibody. DNA was counterstained with 4′,6′-diamino-2-phenylindole (DAPI). Note the displacement over the chromosomal arms when kinase activity is inhibited by NA-PP1.

Article Snippet: Immunoprecipitated or recombinant kinases were included into a 25-μl reaction containing kinase buffer (10 m m MgCl 2 , 25 m m HEPES, pH 7.5, 25 m m β-glycerophosphate, 0.5 m m DTT and 0.5 m m vanadate), 0.2 mg/ml histone H3 (Roche Diagnostics), and 100 μ m ATP with or without 2 μ m NA-PP1 (Calbiochem/Merck Chemicals).

Techniques: Expressing, Blocking Assay, Activity Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: The Aurora B analog-sensitive mutant thiophosphorylates multiple proteins in cell extracts. A, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B, co-purified from SF9 cells, in the presence of ATP and with or without NA-PP1 (2 μm). Histone H3 was used as a substrate. B, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B (wild-type or analog-sensitive mutants) in the presence of ATPγS, N6-furfuryl-ATPγS (Fu), N6-phenylethyl-ATPγS (Phe), or N6-benzyl-ATPγS (Bn). Thiophosphorylated histone H3 was detected with a thiophosphate ester epitope-specific antibody (13). C, in vitro kinase assay with His-INCENP/GST-Aurora BL154G/H250Y in the presence of N6-furfuryl-ATPγS. 50 μg of a mitotic HeLa cell lysate was used as input, and histone H3 was spiked into this lysate to serve as positive control. For the lower part of the blot (containing proteins with molecular masses of 15–50 kDa), a longer exposure (5 min instead of 15 s for the short exposure) is shown.

Article Snippet: Immunoprecipitated or recombinant kinases were included into a 25-μl reaction containing kinase buffer (10 m m MgCl 2 , 25 m m HEPES, pH 7.5, 25 m m β-glycerophosphate, 0.5 m m DTT and 0.5 m m vanadate), 0.2 mg/ml histone H3 (Roche Diagnostics), and 100 μ m ATP with or without 2 μ m NA-PP1 (Calbiochem/Merck Chemicals).

Techniques: Mutagenesis, In Vitro, Kinase Assay, Recombinant, Purification, Positive Control

Journal:

Article Title: Wnt-induced dephosphorylation of Axin releases ?-catenin from the Axin complex

doi:

Figure Lengend Snippet: The Axin protein is phosphorylated. (A) PP2A, but not PP1, dephosphorylates the Axin protein in vitro. Axin protein was immunoprecipitated from cell extracts of C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium. The immune complexes were treated with phosphatase buffer only, PP2A, or PP1 and immunoblotted with anti-Axin antibody. (B) The phosphatase inhibitor okadaic acid inhibits the dephosphorylation of Axin. Cell lysates were prepared from C57MG cells treated with control (−) or Wnt-3A (+)-conditioned medium and with or without 400 nm okadaic acid and immunoblotted with anti-Axin antibody. (C) Lithium, an inhibitor of GSK-3β, promotes the dephosphorylation of Axin. Cell lysates were prepared from C57MG cells first treated with or without 25 mm LiCl for 4 hr and treated with control (−) or Wnt3A (+)-conditioned medium for an additional 2 hr.

Article Snippet: For phosphatase treatment, the immune complexes prior to boiling were incubated in phosphatase reaction buffer (lysis buffer with 0.1% β-mercaptoethanol, 1 m m EGTA, 0.5 mg/ml BSA, and protease inhibitors) and incubated with 1 unit of PP1 (Promega) or 1 unit of PP2A (Upstate Biotechnologies, Inc.) for 1 hr at 30°C.

Techniques: In Vitro, Immunoprecipitation, Control, De-Phosphorylation Assay