Journal: Frontiers in Cardiovascular Medicine

Article Title: Alterations in cytoskeletal and Ca 2+ cycling regulators in atria lacking the obscurin Ig58/59 module

doi: 10.3389/fcvm.2023.1085840

Figure Lengend Snippet: Expression and phosphorylation status of Ig58/59 binding partners and selected Ca 2+ cycling proteins are unaffected in aged Obscn-ΔIg58/59 atria. (A,A’) Representative Coomassie Blue stained agarose gels and immunoblots (A) and relative quantifications (A’) did not reveal statistically significant alterations in the expression or phosphorylation status of giant titins, novex-3, PLN, sAnk1, SLN, SERCA2, or RyR2 in lysates prepared from 6- and 12-month-old Obscn-ΔIg5859 atria; MHC, Hsp90, and GAPDH served as loading controls; n = 3–6 animals per group; data points represent the average of at least two technical replicas; quantification of phosphorylation levels are normalized to total PLN or RyR2 levels; pent, pentamer; mono, monomer.

Article Snippet: The following primary antibodies were used for western blotting: rabbit polyclonal antibodies to obscurin Ig58/59 (1 μg/mL) , obscurin Ig67 (1 μg/mL) , novex-3 (1:1000, a generous gift from Dr. Henk Granzier) , PLN-pSer16 (1:1000; 07–052, Millipore, Temecula, CA, United States), PLN-pThr17 (1:2000; A010-13AP, Badrilla, Leeds, United Kingdom), sAnk1 (1 μg/mL) , RyR2-pSer2808 (1:2000; ab59225, Abcam, Cambridge, MA, United States), RyR2-pSer2814 (1:500; A010-31AP, Badrilla), sarcolipin (1 μg/mL, a generous gift from Dr. Robert Bloch) , ERK2 (1:1000; CST-9108S, Cell Signaling Technology, Danvers, MA, United States), ERK1/2-pThr183/pTyr185 (1:2000; CST-4370 T, Cell Signaling Technology), rabbit monoclonal antibodies to Hsp90 (1:1000; CST-4877, Cell Signaling Technology), and mouse monoclonal antibodies to PLN (1:5000; ab2865, Abcam), SERCA2 (1:1000; MA3-919, Thermo Fisher Scientific, Waltham, MA, United States), RyR2 (1:1000; MA3-925, Thermo Fisher Scientific), GAPDH (1:15000; G8795, Millipore), and α-actinin (1:2500; A7811, Sigma-Aldrich, St. Louis, MO, United States).

Techniques: Expressing, Binding Assay, Staining, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Role of chronic ryanodine receptor phosphorylation in heart failure and ?-adrenergic receptor blockade in mice

doi: 10.1172/JCI37649

Figure Lengend Snippet: (A) Progressive oxidation, Cys-nitrosylation (Cys NO) of cardiac RyR2, and depletion of PDE4D3, calstabin2, PP2A, and PP1 from the cardiac RyR2 complex in the RyR2-S2808D mice. (B) Levels of proteins in the RyR2 complex and levels of oxidation and Cys-nitrosylation were normalized to the total amount of RyR2 (AU). *P < 0.05 versus WT at 1.5 months. (C) Oxidation and Cys-nitrosylation of RyR2 in HF samples. RyR2 immunoprecipitations were performed on hearts from mice with MI and sham-treated mice or on samples from HF and non-HF human heart tissue. (D) Levels of oxidation and Cys-nitrosylation were normalized to the total amount of RyR2 (AU). *P < 0.05 versus normal samples.

Article Snippet: Proteins were transferred to nitrocellulose membranes and immunoblots were developed using the following antibodies: anti-calstabin (1:1,000) ( 55 ), anti-RyR (5029; 1:3,000) ( 55 ), anti-phospho-RyR2-pSer 2808 (1:5,000) ( 49 ), anti-PDE4D3 (1:1,000) ( 16 ), anti-PP2a (1:1,000) (BD Transduction Laboratories), anti-PP1 (1:1,000) (BD Transduction Laboratories), and anti–Cys-NO (1:1,000) (Sigma-Aldrich).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Role of chronic ryanodine receptor phosphorylation in heart failure and ?-adrenergic receptor blockade in mice

doi: 10.1172/JCI37649

Figure Lengend Snippet: (A) Progressive oxidation, Cys-nitrosylation (Cys NO) of cardiac RyR2, and depletion of PDE4D3, calstabin2, PP2A, and PP1 from the cardiac RyR2 complex in the RyR2-S2808D mice. (B) Levels of proteins in the RyR2 complex and levels of oxidation and Cys-nitrosylation were normalized to the total amount of RyR2 (AU). *P < 0.05 versus WT at 1.5 months. (C) Oxidation and Cys-nitrosylation of RyR2 in HF samples. RyR2 immunoprecipitations were performed on hearts from mice with MI and sham-treated mice or on samples from HF and non-HF human heart tissue. (D) Levels of oxidation and Cys-nitrosylation were normalized to the total amount of RyR2 (AU). *P < 0.05 versus normal samples.

Article Snippet: Proteins were transferred to nitrocellulose membranes and immunoblots were developed using the following antibodies: anti-calstabin (1:1,000) ( 55 ), anti-RyR (5029; 1:3,000) ( 55 ), anti-phospho-RyR2-pSer 2808 (1:5,000) ( 49 ), anti-PDE4D3 (1:1,000) ( 16 ), anti-PP2a (1:1,000) (BD Transduction Laboratories), anti-PP1 (1:1,000) (BD Transduction Laboratories), and anti–Cys-NO (1:1,000) (Sigma-Aldrich).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Role of chronic ryanodine receptor phosphorylation in heart failure and ?-adrenergic receptor blockade in mice

doi: 10.1172/JCI37649

Figure Lengend Snippet: (A) Progressive oxidation, Cys-nitrosylation (Cys NO) of cardiac RyR2, and depletion of PDE4D3, calstabin2, PP2A, and PP1 from the cardiac RyR2 complex in the RyR2-S2808D mice. (B) Levels of proteins in the RyR2 complex and levels of oxidation and Cys-nitrosylation were normalized to the total amount of RyR2 (AU). *P < 0.05 versus WT at 1.5 months. (C) Oxidation and Cys-nitrosylation of RyR2 in HF samples. RyR2 immunoprecipitations were performed on hearts from mice with MI and sham-treated mice or on samples from HF and non-HF human heart tissue. (D) Levels of oxidation and Cys-nitrosylation were normalized to the total amount of RyR2 (AU). *P < 0.05 versus normal samples.

Article Snippet: Proteins were transferred to nitrocellulose membranes and immunoblots were developed using the following antibodies: anti-calstabin (1:1,000) ( 55 ), anti-RyR (5029; 1:3,000) ( 55 ), anti-phospho-RyR2-pSer 2808 (1:5,000) ( 49 ), anti-PDE4D3 (1:1,000) ( 16 ), anti-PP2a (1:1,000) (BD Transduction Laboratories), anti-PP1 (1:1,000) (BD Transduction Laboratories), and anti–Cys-NO (1:1,000) (Sigma-Aldrich).

Techniques: