282s 1000  (Genesee Scientific)

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: Impact of oxidants on protein phosphorylation in ARVMs. A, ARVMs were 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). Cell lysates were resolved by SDS-PAGE and gels were stained for phosphoproteins by ProQ-Diamond. Total protein was visualized by SYPRO Ruby and Coomassie staining. B, phosphorylation of cMyBP-C (pSer-273, pSer-282, and Ser-302), PLN (pSer-16), and cTnI (pSer-23/24) in response to oxidant exposure was investigated by immunoblotting (IB) and phospho-specific antibodies. Scatter plots summarize data of at least 7 (phospho-cMyBP-C), 4 (phospho-PLN), or 5 (phospho-cTnI) independent experiments normalized to α-actinin and expressed as % of signal induced by ISO. *, p < 0.05; ***, p < 0.001 for comparison with vehicle control by one-way ANOVA and Dunnett's post-test (F and p values): a, pSer-273 cMyBP-C: F = 79.70, p < 0.0001; b, pSer-282 cMyBP-C: F = 142.4, p < 0.0001; c, pSer-302 cMyBP-C: F = 15.27, p < 0.0001; d, pSer-16 PLB: F = 259.1, p < 0.0001; e, pcTnI: F = 1.246, p = 0.3192). C, ARVMs were treated with the indicated concentrations of NCA (for 30 min), DIA (for 10 min), CXL-1020 (for 15 min), and AS (for 15 min) or were exposed to vehicle (DMSO for 30 min or NaOH for 15 min) or ISO (10 nmol/liter, 10 min). Phosphorylation of cMyBP-C (pSer-282) and PLN (pSer-16) was analyzed by IB using the respective phospho-specific antibodies. Protein loading was assessed using an antibody against α-actinin. Blots are representative of three independent experiments. ns, not significant.

Article Snippet: OA and the anti-pSer-282 cMyBP-C antibody (dilution IB 1:1000; rabbit polyclonal; number ALX-215-057-R050) was from Enzo Life Sciences.

Techniques: SDS Page, 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: 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: OA and the anti-pSer-282 cMyBP-C antibody (dilution IB 1:1000; rabbit polyclonal; number ALX-215-057-R050) was from Enzo Life Sciences.

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 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: OA and the anti-pSer-282 cMyBP-C antibody (dilution IB 1:1000; rabbit polyclonal; number ALX-215-057-R050) was from Enzo Life Sciences.

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