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mouse anti pp1α (Proteintech)

Name: mouse anti pp1α
Brand: Proteintech
Rating: 93 (11 reviews)

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Proteintech mouse anti pp1α
Mouse Anti Pp1α, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti pp1α/product/Proteintech
Average 93 stars, based on 11 article reviews

mouse anti pp1α - by Bioz Stars, 2026-03

93/100 stars

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Fig. 4. SDS22-PP1-I3 disassembly coupled to formation of an active PP1-GADD34 holoenzyme. (A) Model for p97-mediated disassembly of SDS22-PP1-I3 coupled to formation of active PP1-GADD34 that dephosphorylates eIF2α. G-actin is required for PP1-GADD34 activity. (B) Coupled PP1 subunit exchange and eIF2α dephosphorylation reactions with purified proteins as analyzed in PhosTag gels. eIF2α was phosphorylated beforehand by incubation with PERK and reisolated. Indicated protein combinations were incubated for 10 min. Note that eIF2α dephosphorylation required p97-p37 along with GADD34 and G-actin when SDS22-PP1-I3 (SPI) was provided as a source of PP1, and increased with increasing p97 concentrations. Representative gel of three repeats. (C) Quantification of B. eIF2α-P present at the end of the reaction for three experiments was normalized to one ΔGADD34 sample, with eIF2α-P + eIF2α = 100%. Shown are means and SD of the absolute values (n = 3). P values were determined by one-way ANOVA with Šídák’s multiple comparisons test. P values > 0.7 are omitted. (D) p97 activity is required for efficient PP1-GADD34 formation in cells after GADD34 induction. GADD34-GFP expression was induced (DOX) for 5 h in a stable HeLa cell line and cells treated with CB-5083 or vehicle alone for 3 h. GADD34-GFP was immunoprecipitated from lysates and associated PP1 analyzed by western blotting with indicated antibodies. Representative gel of three biological replicates. (E) Quantification of band intensities in D. Mean ± SD from three biological replicates. The P value was determined by the one sample t test.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases.

doi: 10.1073/pnas.2408787121

Figure Lengend Snippet: Fig. 4. SDS22-PP1-I3 disassembly coupled to formation of an active PP1-GADD34 holoenzyme. (A) Model for p97-mediated disassembly of SDS22-PP1-I3 coupled to formation of active PP1-GADD34 that dephosphorylates eIF2α. G-actin is required for PP1-GADD34 activity. (B) Coupled PP1 subunit exchange and eIF2α dephosphorylation reactions with purified proteins as analyzed in PhosTag gels. eIF2α was phosphorylated beforehand by incubation with PERK and reisolated. Indicated protein combinations were incubated for 10 min. Note that eIF2α dephosphorylation required p97-p37 along with GADD34 and G-actin when SDS22-PP1-I3 (SPI) was provided as a source of PP1, and increased with increasing p97 concentrations. Representative gel of three repeats. (C) Quantification of B. eIF2α-P present at the end of the reaction for three experiments was normalized to one ΔGADD34 sample, with eIF2α-P + eIF2α = 100%. Shown are means and SD of the absolute values (n = 3). P values were determined by one-way ANOVA with Šídák’s multiple comparisons test. P values > 0.7 are omitted. (D) p97 activity is required for efficient PP1-GADD34 formation in cells after GADD34 induction. GADD34-GFP expression was induced (DOX) for 5 h in a stable HeLa cell line and cells treated with CB-5083 or vehicle alone for 3 h. GADD34-GFP was immunoprecipitated from lysates and associated PP1 analyzed by western blotting with indicated antibodies. Representative gel of three biological replicates. (E) Quantification of band intensities in D. Mean ± SD from three biological replicates. The P value was determined by the one sample t test.

Article Snippet: Rabbit polyclonal anti- NIPP1 (Sigma Cat. no. HPA027452, 1:500), goat polyclonal anti- SDS22 (Santa Cruz E- 20, Cat. no. sc- 162164), mouse monoclonal anti- SDS22 (B- 6) (Santa Cruz, Cat. no. sc- 398864, 1:1,000), rabbit polyclonal anti- I3 [(34), 1:1,000], mouse monoclonal anti- GFP (Roche, Cat. no. 11814460001, 1:500), mouse monoclonal anti- PP1α (E- 9) (Santa Cruz C- 19, Cat. no. sc- 7482, 1:500), mouse monoclonal anti- PP1γ (Santa Cruz E- 4, Cat. no. sc- 515943, 1:1,000), goat polyclonal anti- PP1γ (Santa Cruz C- 19, Cat. no. sc- 6108 1:500), rabbit polyclonal anti- KNL1 [(35), 1:750], rabbit polyclonal anti- URI1 (New England Biolabs, Cat. no. 5844, 1:1,000), mouse monoclonal anti- puromycin (Sigma Cat. no. MABE343, 1:20,000), rabbit polyclonal anti- PPP1R15B (CReP) (Proteintech Cat. no. 14634- 1- AP, 1:2,000), mouse monoclonal anti- α- tubulin (Sigma Cat. no. T- 5168, 1:10,000).

Techniques: Activity Assay, De-Phosphorylation Assay, Purification, Incubation, Expressing, Immunoprecipitation, Western Blot

Fig. 5. Model for regulation of PP1 by cycling between SDS22 and I3 binding and release. The SDS22-PP1-I3 complex serves as a thermodynamic sink for free PP1, with SDS22 and I3 even displacing holoenzyme subunits. Once formed, p97 consumes ATP to disassemble the SDS22-PP1-I3 complex. PP1 is thus made available for formation of active holoenzymes and substrate dephosphorylation. When p97 is compromised, PP1 is sequestered in the inactive SDS22-PP1-I3 complex. This cycle protects cells from free PP1 and ensures a dynamic formation of holophosphatase complexes.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases.

doi: 10.1073/pnas.2408787121

Figure Lengend Snippet: Fig. 5. Model for regulation of PP1 by cycling between SDS22 and I3 binding and release. The SDS22-PP1-I3 complex serves as a thermodynamic sink for free PP1, with SDS22 and I3 even displacing holoenzyme subunits. Once formed, p97 consumes ATP to disassemble the SDS22-PP1-I3 complex. PP1 is thus made available for formation of active holoenzymes and substrate dephosphorylation. When p97 is compromised, PP1 is sequestered in the inactive SDS22-PP1-I3 complex. This cycle protects cells from free PP1 and ensures a dynamic formation of holophosphatase complexes.

Techniques: Binding Assay, De-Phosphorylation Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Figure Lengend Snippet: Full pancreas proteomics

Article Snippet: Immunoblots were probed with goat anti-spinophilin antibody, mouse anti-myosin-9 antibody (MilliporeSigma MABT164), mouse anti-neurabin antibody (Santa Cruz Biotechnology, SC-136327), or mouse anti-PP1α antibody (Santa Cruz Biotechnology, SC-7482) and infrared secondary antibodies (From Jackson ImmunoResearch or Invitrogen) and developed on a Li-Cor Odyssey or Odyssey M (Li-Cor Biosciences, Lincoln, NE).

Techniques:

Loss of spinophilin significantly reduces weight gain in lean and two mouse models of obesity. A and B : male ( A ) or female ( B ) Lepr +/+ / Spino +/+ , Lepr db/db / Spino +/+ , Lepr +/+ / Spino −/− , and Lepr db/db / Spino −/− mice weights were taken bi-weekly from 4 to 20 wk and plotted. A mixed-effects three-way ANOVA was performed initially, followed by a two-way ANOVA to determine the effect of spinophilin genotype, age, and an interaction within the Lepr +/+ and Lepr db/db genotypes individually. C and D : male ( C ) and female ( D ) Spino +/+ /high-fat diet-fed (HFF) and Spino −/− /HFF mice were weighed bi-weekly and plotted. For two-way ANOVAs, significant spinophilin genotype, time, and interaction (spinophilin genotype × time) are shown. n = 4–12 mice per group for each age point. Data ± SE are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Figure Lengend Snippet: Loss of spinophilin significantly reduces weight gain in lean and two mouse models of obesity. A and B : male ( A ) or female ( B ) Lepr +/+ / Spino +/+ , Lepr db/db / Spino +/+ , Lepr +/+ / Spino −/− , and Lepr db/db / Spino −/− mice weights were taken bi-weekly from 4 to 20 wk and plotted. A mixed-effects three-way ANOVA was performed initially, followed by a two-way ANOVA to determine the effect of spinophilin genotype, age, and an interaction within the Lepr +/+ and Lepr db/db genotypes individually. C and D : male ( C ) and female ( D ) Spino +/+ /high-fat diet-fed (HFF) and Spino −/− /HFF mice were weighed bi-weekly and plotted. For two-way ANOVAs, significant spinophilin genotype, time, and interaction (spinophilin genotype × time) are shown. n = 4–12 mice per group for each age point. Data ± SE are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Techniques:

Spino −/− mice have improved glucose tolerance test (GTT) in high-fat diet-fed (HFF) male and female mice. A : intraperitoneal glucose tolerance test (IPGTT) of HFF wild-type (WT) and spinophilin knockout (KO) male mice at 6 wk of age. B : area under the curve for the IPGTT from HFF WT and spinophilin KO male mice at 6 wk of age. C : weights from male HFF WT and spinophilin KO mice at 6 wk of age. D : IPGTT of HFF WT and spinophilin KO female mice at 6 wk of age. E : area under the curve for the IPGTT from HFF WT and spinophilin KO female mice at 6 wk of age. F : weights from female HFF WT and spinophilin KO mice at 6 wk of age. G : IPGTT of HFF WT and spinophilin KO male mice at 10 wk of age. H : area under the curve for the IPGTT from HFF WT and spinophilin KO male mice at 10 wk of age. I : weights from male HFF WT and spinophilin KO mice at 10 wk of age. J : IPGTT of HFF WT and spinophilin KO female mice at 10 wk of age. K : area under the curve for the IPGTT from HFF WT and spinophilin KO female mice at 10 wk of age. L : weights from female HFF WT and spinophilin KO mice at 10 wk of age. Data are given with SE. A two-way repeated-measures ANOVA ( A , D , G , and J ) or unpaired t tests ( B , C , E , F , H , and I ) were performed. For two-way ANOVA, significant spinophilin genotype, time, and interaction (spinophilin genotype × time) are shown. n = 5–6 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Figure Lengend Snippet: Spino −/− mice have improved glucose tolerance test (GTT) in high-fat diet-fed (HFF) male and female mice. A : intraperitoneal glucose tolerance test (IPGTT) of HFF wild-type (WT) and spinophilin knockout (KO) male mice at 6 wk of age. B : area under the curve for the IPGTT from HFF WT and spinophilin KO male mice at 6 wk of age. C : weights from male HFF WT and spinophilin KO mice at 6 wk of age. D : IPGTT of HFF WT and spinophilin KO female mice at 6 wk of age. E : area under the curve for the IPGTT from HFF WT and spinophilin KO female mice at 6 wk of age. F : weights from female HFF WT and spinophilin KO mice at 6 wk of age. G : IPGTT of HFF WT and spinophilin KO male mice at 10 wk of age. H : area under the curve for the IPGTT from HFF WT and spinophilin KO male mice at 10 wk of age. I : weights from male HFF WT and spinophilin KO mice at 10 wk of age. J : IPGTT of HFF WT and spinophilin KO female mice at 10 wk of age. K : area under the curve for the IPGTT from HFF WT and spinophilin KO female mice at 10 wk of age. L : weights from female HFF WT and spinophilin KO mice at 10 wk of age. Data are given with SE. A two-way repeated-measures ANOVA ( A , D , G , and J ) or unpaired t tests ( B , C , E , F , H , and I ) were performed. For two-way ANOVA, significant spinophilin genotype, time, and interaction (spinophilin genotype × time) are shown. n = 5–6 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Techniques: Knock-Out

Spinophilin protein interactions with protein phosphatase 1 (PP1), neurabin, and myosin-9 in wild-type (WT), Ins2 Akita , and Lepr db/db mice. A : spinophilin was immunoprecipitated from pancreas of adult WT, Ins2 Akita (AK), and Lepr db/db (DB) mice. Immunoblotting for spinophilin, PP1α, myosin-9, and neurabin was performed. B and C : PP1α ( B ) or neurabin ( C ) expression in spinophilin immunoprecipitates was normalized to spinophilin expression in the immuno precipitate. A normalized ratio was plotted. n = 3–5 mice per group. ** P < 0.01. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Figure Lengend Snippet: Spinophilin protein interactions with protein phosphatase 1 (PP1), neurabin, and myosin-9 in wild-type (WT), Ins2 Akita , and Lepr db/db mice. A : spinophilin was immunoprecipitated from pancreas of adult WT, Ins2 Akita (AK), and Lepr db/db (DB) mice. Immunoblotting for spinophilin, PP1α, myosin-9, and neurabin was performed. B and C : PP1α ( B ) or neurabin ( C ) expression in spinophilin immunoprecipitates was normalized to spinophilin expression in the immuno precipitate. A normalized ratio was plotted. n = 3–5 mice per group. ** P < 0.01. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Techniques: Immunoprecipitation, Western Blot, Expressing

Loss of spinophilin, specifically in β cells, improves glucose tolerance and reduces insulin tolerance. A : glucose tolerance test (GTT) of 6-wk-old β cell-specific spinophilin knockout (KO) mice (Spino ΔIns ) with Cre and flox controls. B : area under the curve for GTT of 6-wk-old Spino ΔIns . C : weights for 6-wk-old Spino ΔIns and controls. D : insulin tolerance test (ITT) (normalized to baseline) of 8-wk-old Spino ΔIns and controls. E : area over the curve for ITT (normalized to baseline) of 8-wk-old Spino ΔIns and controls. F : weights for 8-wk-old Spino ΔIns and controls. G : GTT of 10-wk-old Spino ΔIns and controls. H : area under the curve for GTT of 10-wk-old Spino ΔIns . I : weights for 10-wk-old Spino ΔIns and controls. T tests or two-way ANOVAs (comparing genotype, time, or a genotype × time interaction) were performed. A Grubbs’s test was performed and identified two outliers, one 10-wk-old Cre control and one 6-wk-old Spino ΔIns mouse, which are not included in these data. n = 3–7 mice per group. * P < 0.05, ** P < 0.01, **** P < 0.001. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Figure Lengend Snippet: Loss of spinophilin, specifically in β cells, improves glucose tolerance and reduces insulin tolerance. A : glucose tolerance test (GTT) of 6-wk-old β cell-specific spinophilin knockout (KO) mice (Spino ΔIns ) with Cre and flox controls. B : area under the curve for GTT of 6-wk-old Spino ΔIns . C : weights for 6-wk-old Spino ΔIns and controls. D : insulin tolerance test (ITT) (normalized to baseline) of 8-wk-old Spino ΔIns and controls. E : area over the curve for ITT (normalized to baseline) of 8-wk-old Spino ΔIns and controls. F : weights for 8-wk-old Spino ΔIns and controls. G : GTT of 10-wk-old Spino ΔIns and controls. H : area under the curve for GTT of 10-wk-old Spino ΔIns . I : weights for 10-wk-old Spino ΔIns and controls. T tests or two-way ANOVAs (comparing genotype, time, or a genotype × time interaction) were performed. A Grubbs’s test was performed and identified two outliers, one 10-wk-old Cre control and one 6-wk-old Spino ΔIns mouse, which are not included in these data. n = 3–7 mice per group. * P < 0.05, ** P < 0.01, **** P < 0.001. All statistics are shown in Supplemental Material ( https://doi.org/10.6084/m9.figshare.22507135.v1 ).

Techniques: Knock-Out, Control

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Figure Lengend Snippet: Full pancreas proteomics

Techniques:

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Mechanisms of spinophilin-dependent pancreas dysregulation in obesity

doi: 10.1152/ajpendo.00099.2023

Techniques: Immunoprecipitation, Western Blot, Expressing

( 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.

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: Samples were immunoblotted using standard techniques and probed using a mouse monoclonal anti-NCC antibody , an anti–p-NCC (T58) antibody , a mouse monoclonal anti-PP1α antibody (Thermo Fisher Scientific, catalog 43-8100), and polyclonal anti–proteasome 20S antibodies (Santa Cruz Biotechnology, catalog sc-67339).

Techniques: Western Blot, Control, Quantitative Proteomics, Labeling

( 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.

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.

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

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