pp2a dimer  (Sino Biological)

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: PLoS ONE

Article Title: Phosphatase-Dependent Regulation of Epithelial Mitogen-Activated Protein Kinase Responses to Toxin-Induced Membrane Pores

doi: 10.1371/journal.pone.0008076

Figure Lengend Snippet: (A)A549 cells were transfected with 2 µg PP1 or scrambled siRNA per 1×10 6 cells and stimulated 54 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP1 siRNA leads to reduced PP1 expression and a corresponding increase in Ply-induced p38 and JNK phosphorylation. (B) A549 cells were transfected with 6 µg PP2Aα/β or control shRNA per 1×10 6 cells and stimulated 60 hrs post-transfection with 100 ng/ml of purified Ply. Transfection with PP2Aα/β shRNA leads to reduced PP2A expression and a moderate increase in p38 phosphorylation.

Article Snippet: PP1 and scrambled siRNAs were from Santa Cruz Biotechnology (Santa Cruz, CA), and PP2Aα/β and GFP control shRNA were constructed by William Hahn and obtained from Addgene (Cambridge, MA) (Addgene plasmids 10676, 15249, and 15250) .

Techniques: Transfection, Purification, Expressing, shRNA