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: 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