human pp2a c santa cruz biotechnol  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Time-dependent impact of NCA on PKA-RI dimerization and protein translocation in ARVMs. A, ARVMs were treated with vehicle for 100 min (0 min NCA), NCA (100 µmol/liter) for 3, 10, 30, and 100 min, or ISO (10 nmol/liter) for 10 min. PKA-RI and cMyBP-C phosphorylation at Ser-282 were detected in whole lysates by IB analysis under NR or R conditions, respectively. Scatter plots summarize RI dimer signal fold-change compared with control sample (0 min NCA) and cMyBP-C phosphorylation as % of ISO-response from 4 independent experiments. Band intensities were normalized to α-actinin. *, p < 0.05; **, p < 0.01; ***, p < 0.001 for comparison with vehicle control (0 min NCA) by one-way ANOVA with Dunnett's post-test (RI dimer: F = 8.23, p = 0.0003; pSer-282 cMyBP-C: F = 107.5, p < 0.0001). B, ARVMs were exposed to vehicle for 100 min (0 min NCA), NCA (100 µmol/liter) for 3, 10, 30, and 100 min or ISO (10 nmol/liter) for 10 min and subcellular fractionation was performed under NR conditions. Phosphorylation of cMyBP-C at Ser-282, as well as the presence of PKA-RI, PKA-C, B56α, and PP2A-C were detected in input lysates and Triton-insoluble fractions. Phosphorylation of cMyBP-C at Ser-282 (n = 6) normalized to Coomassie stain signals (not shown) is presented as % of the band intensity induced by ISO. PKA-RI (n = 4), PKA-C (n = 6), B56α (n = 5), and PP2A-C (n = 6) signals from Triton-insoluble fractions normalized to the corresponding inputs are shown as fold-change of the control signal (0 min NCA). *, p < 0.05; **, p < 0.01; ***, p < 0.001 for comparison with the corresponding vehicle control (sample 0 min) by one-way ANOVA with Dunnett's post-test (F and p values): a, PKA-RI, F = 15.18, p < 0.0001; b, PKA-C, F = 15.61, p < 0.0001; c, B56α, F = 4.78, p = 0.0036; d, PP2A-C, F = 9.16, p < 0.0001; e, pSer-282 cMyBP-C, F = 67.33, p < 0.0001). ns, not significant.

Article Snippet: Recombinant human PP2A-C (number 10011237; lot 0521439-1), AS and ODQ were purchased from Cayman Chemicals (Ann Arbor, MI USA).

Techniques: Translocation Assay, Fractionation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Oxidation-mediated effects of NCA on PKA and PP2A-C translocation. Following incubation of ARVMs with vehicle (control), NCA (100 µmol/liter, 30 min), or ISO (10 nmol/liter, 10 min), cells were harvested under NR or R conditions and separated into input Triton-soluble and Triton-insoluble fractions. cTnI was employed as the Triton-insoluble fraction marker protein. The content of PKA-RI, PKA-C, B56α, and PP2A-C in input lysates and in the Triton-insoluble fractions from both harvesting conditions was compared in 3 independent experiments. Signals obtained from Triton-insoluble samples were normalized to the related inputs and are presented as fold-change of vehicle control (R) in the scatter plots. **, p < 0.01; ***, p < 0.001 for comparison with the corresponding vehicle control; #, p < 0.05 for comparison between similar treatments from different harvesting conditions by two-way ANOVA with Bonferroni post-test (F and P values): a, PKA-RI, interaction, F = 4.24, p = 0.0403; treatment, F = 6.61, p = 0.0116; harvesting conditions, F = 3.91, p = 0.0714; b, PKA-C, interaction, F = 6.30, p = 0.0135; treatment, F = 11.03, p = 0.0019; harvesting conditions, F = 0.2196, p = 0.6477; c, B56α, interaction, F = 2.93, p = 0.0917; treatment,, F = 9.66, p = 0.0032; harvesting conditions, F = 5.89, p = 0.0320; d, PP2A-C, interaction, F = 4.23, p = 0.0406; treatment, F = 8.35, p = 0.0053; harvesting conditions, F = 9.86, p = 0.0085).

Article Snippet: Recombinant human PP2A-C (number 10011237; lot 0521439-1), AS and ODQ were purchased from Cayman Chemicals (Ann Arbor, MI USA).

Techniques: Translocation Assay, Incubation, Marker

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Investigation of PKA and PP2A oxidation in intact ARVMs. A, biotin-switch analysis. Lysates from ARVMs exposed to vehicle (DMSO; 30 min) or NCA (100 µmol/liter; 30 min) were subjected to biotin labeling of oxidized thiol groups and subsequently biotinylated proteins were enriched by streptavidin pulldown. Total protein content before biotin labeling (Input; Lysate) or before streptavidin-pulldown (Input; Biotin) is demonstrated by Coomassie (left panel) or SYPRO Ruby staining (middle panel). Protein biotinylation levels correlated with protein oxidation in samples before (Input; Biotin) and after streptavidin-pulldown (eluate) was visualized by chemiluminescent detection with streptavidin-HRP (right panel). Blots are representative of three independent experiments. B, PKA-RI, PKA-C, B56α, and PP2A-C were detected in the streptavidin-pulldown samples by Western immunoblot analysis (IB). C, PEG-switch analysis. Lysates from ARVMs exposed to vehicle (control), NCA (100 µmol/liter, 30 min), AS (300 µmol/liter, 15 min), CXL-1020 (300 µmol/liter, 15 min), H2O2 (100 µmol/liter, 10 min), DIA (500 µmol/liter, 10 min), or ISO (10 nmol/liter, 10 min) were analyzed by nonreducing (left panel) or reducing (middle panel) Western immunoblot analysis for PKA-RI, PKA-C, B56α, and PP2A-C. Lysates were subjected to PEG tag labeling of oxidized thiol groups and subsequent detection of oxidized proteins via a mass shift was performed by western immunoblotting for PKA-RI, PKA-C, B56α, and PP2A-C (right panel). Blots are representative of three independent experiments.

Article Snippet: Recombinant human PP2A-C (number 10011237; lot 0521439-1), AS and ODQ were purchased from Cayman Chemicals (Ann Arbor, MI USA).

Techniques: Labeling, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Oxidation-mediated effects on PKA and PP2A-C activity in vitro. A, active PKA catalytic subunit (PKA-C) was preincubated for 10 min with either ATP (100 µmol/liter), H89 (25 µmol/liter), or assay buffer (Ø) and then exposed to vehicle (control) or 100 µmol/liter NCA for 30 min. Then, the samples that initially did not contain ATP were supplemented with 100 µmol/liter of ATP and the in vitro kinase reaction was initiated by adding 100 pmol of recombinant His6-tagged C1-M-C2 cMyBP-C to each sample. Samples that did not contain kinase served as additional controls. Substrate phosphorylation and content was assessed under R conditions (sample reduction with 10% (v/v) β-mercaptoethanol) by IB using antibodies against pSer-282-cMyBP-C and the His6 tag. The presence and oxidation of PKA-C was monitored under NR conditions by IB using an anti-PKA-C antibody. Coomassie stain of the substrate blot is also shown as additional loading reference. The scatter plot summarizes results for C1-M-C2 phosphorylation from 4 independent experiments. Data are expressed as % of the signal of the vehicle control after ATP preincubation. **, p < 0.01; ***, p < 0.001 for comparison with the corresponding assay buffer (Ø)-pretreated sample; #, p < 0.05; ###, p < 0.001 for comparison with corresponding samples with and without NCA treatment by two-way ANOVA with Bonferroni post-test (interaction, F = 7.07, p = 0.0054; preincubation buffer, F = 31.64, p < 0.0001; treatment: F = 22.50, p = 0.0002). B, active recombinant human PP2A-C was incubated with vehicle (DMSO), NCA (100 μmol/liter), CXL-1020 (300 μmol/liter), or OA (10 nmol/liter), and the substrate DiFMUP (500 µmol/liter). Fluorescence reflecting phosphatase activity was monitored over time (representative traces). For each treatment, a phosphatase-free blank sample was included in the measurement. The table shows the activity of active recombinant PP2A-C at each condition, calculated from the slope of the linear regression of averaged RFU values and expressed as ΔRFU/min or % of the activity calculated under control conditions (n = 4-5 experiments). Measured fluorescence was normalized to blank values and expressed as % of the control signal. C, the data from B were reanalyzed to reflect the HNO donor incubation times in cell culture experiments. The scatter plots represent average phosphatase activity expressed as ΔRFU/min calculated from the slope of a linear curve fit after 15 min of CXL-1020 or 30 min of NCA treatment (n = 4-5 experiments) to reflect the cell culture conditions. *, p < 0.05; **, p < 0.01 two-tailed Student's t test for comparison of each time point with the corresponding vehicle control; ns, not significant.

Article Snippet: Recombinant human PP2A-C (number 10011237; lot 0521439-1), AS and ODQ were purchased from Cayman Chemicals (Ann Arbor, MI USA).

Techniques: Activity Assay, In Vitro, Recombinant, Staining, Incubation, Fluorescence, Cell Culture, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Oxidation-mediated effects on PKA and PP2A-C activity in myofilaments. A, Triton X-100–insoluble myofilament-containing fractions were prepared under NR or R conditions from ARVMs after exposure to NCA. Samples were split and in vitro phosphorylation of His6-tagged C1-M-C2 cMyBP-C at Ser-282 analyzed after addition of vehicle (DMSO), DTT to reduce oxidative modifications, or OA to inhibit PP1α and PP2A. Equal substrate protein content was demonstrated by immunoblotting with an anti-His antibody. The scatter plot summarizes results for C1-M-C2 phosphorylation from 3 to 4 independent experiments. Data are expressed as % of the signal of the NR signal without any additions. *, p < 0.05 for comparison with the NR nontreated sample; ####, p < 0.001 for comparison of the NR + OA sample in the presence or absence of DTT. Analysis by two-way ANOVA with Bonferroni post-test (interaction, F = 7.39, p = 0.0013; harvesting conditions, F = 36.99, p < 0.0001; IVK conditions, F = 14.22, p < 0.0001). B, Triton X-100–insoluble myofilament-containing fractions were prepared under NR or R conditions from ARVMs after exposure to NCA. Samples were split and phosphatase activity over time recorded after addition of vehicle (PBS) or DTT and DiFMUP (representative traces). The bar chart summarizes the results of 3 to 4 independent experiments expressed as % of the phosphatase activity measured under NR condition without any addition. ***, p < 0.001 for comparison with the NR DTT-treated sample. Analysis by two-way ANOVA with Bonferroni post-test (interaction, F = 10.14, p = 0.0097; harvesting conditions, F = 51,53, p < 0.0001; phosphatase assay conditions, F = 27,01, p < 0.0004). ns, nonsignificant.

Article Snippet: Recombinant human PP2A-C (number 10011237; lot 0521439-1), AS and ODQ were purchased from Cayman Chemicals (Ann Arbor, MI USA).

Techniques: Activity Assay, In Vitro, Western Blot, Phosphatase Assay

Journal: The Journal of Biological Chemistry

Article Title: Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

doi: 10.1074/jbc.RA120.014467

Figure Lengend Snippet: Summary scheme. Effect of HNO donor-mediated oxidation on kinase (PKA; left) and phosphatase (PP2A; right) signaling in adult rat ventricular myocytes. RI, regulatory type I PKA subunit; C, catalytic subunit of PKA (left) or PP2A (right); A, regulatory subunit; B, targeting subunit; P, protein phosphorylation.

Article Snippet: Recombinant human PP2A-C (number 10011237; lot 0521439-1), AS and ODQ were purchased from Cayman Chemicals (Ann Arbor, MI USA).

Techniques:

Journal: Journal of neuroscience research

Article Title: Hindbrain A2 Noradrenergic Neuron Adenosine 5′-Monophosphate-Activated Protein Kinase (AMPK) Activation, Upstream Kinase/Phosphorylase Protein Expression, and Receptivity to Hormone and Fuel Reporters of Short-Term Food Deprivation are Regulated by Estradiol

doi: 10.1002/jnr.23892

Figure Lengend Snippet: Description of Primary Antibodies Used

Article Snippet: ; rabbit; sc-15318; Lot no. B1413; AB_2089347 0.4 ug/mL PP2A-C alpha/beta (7A6) Unmethylated C-terminus amino acids 302–309 of human PP2A-C Santa Cruz Biotechnol.

Techniques: Concentration Assay

Journal: Journal of neuroscience research

Article Title: Hindbrain A2 Noradrenergic Neuron Adenosine 5′-Monophosphate-Activated Protein Kinase (AMPK) Activation, Upstream Kinase/Phosphorylase Protein Expression, and Receptivity to Hormone and Fuel Reporters of Short-Term Food Deprivation are Regulated by Estradiol

doi: 10.1002/jnr.23892

Figure Lengend Snippet: Description of Primary Antibodies Used

Article Snippet: PP2A-C alpha/beta (7A6) , Unmethylated C-terminus amino acids 302–309 of human PP2A-C , Santa Cruz Biotechnol.; mouse; sc-81601; Lot no. J1410; AB_1128773 , 0.4 ug/mL.

Techniques: Concentration Assay