Journal: Frontiers in Physiology

Article Title: Acetate suppresses myocardial contraction via the short-chain fatty acid receptor GPR43

doi: 10.3389/fphys.2022.1111156

Figure Lengend Snippet: GLPG0974 prevented the acetate-induced inhibition of myocardial contraction. Photomicrographs showing GPR43 and RyR2 were co-expressed in myocardial cell (A) , GPR43 was labeled red, RyR2 was labeled red green and DAPI was labeled blue in the nucleus; scale bar, 20 μm. The GPR43 protein expression in myocardial cells was observed by Western blot (B) . Representative traces of myocardial contraction in isolated myocardial cells (C) . The sarcomere contraction amplitude (D) , diastolic sarcomere length (E) , maximum contraction velocity (F) , and maximum relaxation velocity (G) in the isolated myocardial cell. N = 5, n = 11. N: the number of rats, n: the number of cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking with 10% normal goat serum (Solarbio, Beijing, China), cells were incubated with GPR43 (dilution 1:100, Cat# AFR-032, RRID: AB_2756592, Alomone labs, Israel) and the type 2 ryanodine receptor (RyR2, dilution 1:100, Cat# ab2827, RRID: AB_2183052, Abcam, United States) antibodies for 12 h in 4°C.

Techniques: Inhibition, Labeling, Expressing, Western Blot, Isolation

Journal: Cardiovascular Diabetology

Article Title: Dapagliflozin reduces the vulnerability of rats with pulmonary arterial hypertension-induced right heart failure to ventricular arrhythmia by restoring calcium handling

doi: 10.1186/s12933-022-01614-5

Figure Lengend Snippet: Western blot analysis. Total RyR2 (t-RyR2), phosphorylated RyR2 (p-RyR2-Ser2814), SERCA2a, total CaMKII (t-CaMKII), p-CaMKII (P-CaMKII-Thr287), phospholamban (PLB), Cav1.2, and Na + –Ca 2+ exchanger protein (NCX) levels in the RV tissue from the 4 groups of rats were analysed and normalised to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). N = 5 per group. The horizontal lines show the mean ± SEM. One-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The protein expression of total ryanodine receptor 2 (t-RyR2) (Affinity, AF0015, 1:500), Ser2814-phosphorylated RyR2 (p-RyR2) (Badrilla, A010-31AP, 1:300), sarco-/endoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a) (Abcam, Ab150435, 1:1000), phospholamban (PLB) (Abcam, Ab85146, 1:1000), total CaMKII (t-CaMKII) (Abcam, Ab134041, 1:1000), Thr287-phosphorylated CaMKII (p-CaMKII) (Thermo Fisher, PA5-37833, 1:500), Cav 1.2 (Abcam, Ab58552, 1:500) and Na + -Ca 2+ exchanger (NCX) (Invitrogen, MA3-926, 1:500) was determined via Western blotting and was normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, ab181602, 1:10000) expression, as previously described [ , ].

Techniques: Western Blot

Journal: Cell reports

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1016/j.celrep.2022.111203

Figure Lengend Snippet: PKA pathway phosphoproteomic changes in STK25 −/− compared with wild type

Article Snippet: For Western blotting antibodies include: HRP conjugated anti-GAPDH (Cell Signaling Technology Cat# 3683, RRID:AB_1642205), anti-STK25 (Abcam Cat# ab157188, RRID:AB_2725788), anti-Flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GM130 (Cell Signaling Technology Cat# 12480, RRID:AB_2797933), anti-PRKAR1A (Abcam Cat# ab139695, RRID:AB_2893184), anti-pS77 PRKAR1A (Abcam Cat#ab139682, RRID:AB_2904566), anti-pS83 PRKAR1A (Abcam Cat#ab154851, RRID:AB_2904567), anti-PRKAR2A (ProteinTech Cat# 10142-2-AP), anti-phospholamban (Cell Signaling Technology Cat# 14562, RRID:AB_2798511), anti-phospho-phospholamban -pS16/T17 (Cell Signaling Technology Cat# 8496, RRID:AB_10949102), anti-Ryanodine receptor 2 (Abcam Cat#ab196355, RRID:AB_2904568), anti-p-Ryanodine receptor 2-pS2808 (Abcam Cat# ab59225, RRID:AB_946327), anti-TnI (Cell Signaling Cat# 4002), anti-TnI-pS23/S24 (Cell Signaling Cat# 4004), anti-V5 (Sigma-Aldrich Cat# V8137, RRID:AB_261889), anti-PKA Catalytic subunit (Abcam Cat# ab26322, RRID:AB_2170049), HRP-conjugated anti-mouse (Cell Signaling Technology Cat# 7076, RRID:AB_330924) and HRP-conjugated anti-rabbit (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) were used for detection.

Techniques: Expressing

Journal: Cell reports

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1016/j.celrep.2022.111203

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For Western blotting antibodies include: HRP conjugated anti-GAPDH (Cell Signaling Technology Cat# 3683, RRID:AB_1642205), anti-STK25 (Abcam Cat# ab157188, RRID:AB_2725788), anti-Flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GM130 (Cell Signaling Technology Cat# 12480, RRID:AB_2797933), anti-PRKAR1A (Abcam Cat# ab139695, RRID:AB_2893184), anti-pS77 PRKAR1A (Abcam Cat#ab139682, RRID:AB_2904566), anti-pS83 PRKAR1A (Abcam Cat#ab154851, RRID:AB_2904567), anti-PRKAR2A (ProteinTech Cat# 10142-2-AP), anti-phospholamban (Cell Signaling Technology Cat# 14562, RRID:AB_2798511), anti-phospho-phospholamban -pS16/T17 (Cell Signaling Technology Cat# 8496, RRID:AB_10949102), anti-Ryanodine receptor 2 (Abcam Cat#ab196355, RRID:AB_2904568), anti-p-Ryanodine receptor 2-pS2808 (Abcam Cat# ab59225, RRID:AB_946327), anti-TnI (Cell Signaling Cat# 4002), anti-TnI-pS23/S24 (Cell Signaling Cat# 4004), anti-V5 (Sigma-Aldrich Cat# V8137, RRID:AB_261889), anti-PKA Catalytic subunit (Abcam Cat# ab26322, RRID:AB_2170049), HRP-conjugated anti-mouse (Cell Signaling Technology Cat# 7076, RRID:AB_330924) and HRP-conjugated anti-rabbit (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) were used for detection.

Techniques: Recombinant, SYBR Green Assay, In Vitro, Activity Assay, Viability Assay, Sequencing, esiRNA, CRISPR, Knock-Out, Plasmid Preparation, Software

Journal: Cell reports

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1016/j.celrep.2022.111203

Figure Lengend Snippet: PKA pathway phosphoproteomic changes in STK25 −/− compared with wild type

Article Snippet: Anti-Ryanodine Receptor 2-pS2808 , Abcam , Cat# ab59225, RRID:AB_946327.

Techniques: Expressing

Journal: Cell reports

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1016/j.celrep.2022.111203

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Ryanodine Receptor 2-pS2808 , Abcam , Cat# ab59225, RRID:AB_946327.

Techniques: Recombinant, SYBR Green Assay, In Vitro, Activity Assay, Viability Assay, Sequencing, esiRNA, CRISPR, Knock-Out, Plasmid Preparation, Software

Journal: Cell reports

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1016/j.celrep.2022.111203

Figure Lengend Snippet: PKA pathway phosphoproteomic changes in STK25 −/− compared with wild type

Article Snippet: For Western blotting antibodies include: HRP conjugated anti-GAPDH (Cell Signaling Technology Cat# 3683, RRID:AB_1642205), anti-STK25 (Abcam Cat# ab157188, RRID:AB_2725788), anti-Flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GM130 (Cell Signaling Technology Cat# 12480, RRID:AB_2797933), anti-PRKAR1A (Abcam Cat# ab139695, RRID:AB_2893184), anti-pS77 PRKAR1A (Abcam Cat#ab139682, RRID:AB_2904566), anti-pS83 PRKAR1A (Abcam Cat#ab154851, RRID:AB_2904567), anti-PRKAR2A (ProteinTech Cat# 10142-2-AP), anti-phospholamban (Cell Signaling Technology Cat# 14562, RRID:AB_2798511), anti-phospho-phospholamban -pS16/T17 (Cell Signaling Technology Cat# 8496, RRID:AB_10949102), anti-Ryanodine receptor 2 (Abcam Cat#ab196355, RRID:AB_2904568), anti-p-Ryanodine receptor 2-pS2808 (Abcam Cat# ab59225, RRID:AB_946327), anti-TnI (Cell Signaling Cat# 4002), anti-TnI-pS23/S24 (Cell Signaling Cat# 4004), anti-V5 (Sigma-Aldrich Cat# V8137, RRID:AB_261889), anti-PKA Catalytic subunit (Abcam Cat# ab26322, RRID:AB_2170049), HRP-conjugated anti-mouse (Cell Signaling Technology Cat# 7076, RRID:AB_330924) and HRP-conjugated anti-rabbit (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) were used for detection.

Techniques: Expressing

Journal: Cell reports

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1016/j.celrep.2022.111203

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For Western blotting antibodies include: HRP conjugated anti-GAPDH (Cell Signaling Technology Cat# 3683, RRID:AB_1642205), anti-STK25 (Abcam Cat# ab157188, RRID:AB_2725788), anti-Flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GM130 (Cell Signaling Technology Cat# 12480, RRID:AB_2797933), anti-PRKAR1A (Abcam Cat# ab139695, RRID:AB_2893184), anti-pS77 PRKAR1A (Abcam Cat#ab139682, RRID:AB_2904566), anti-pS83 PRKAR1A (Abcam Cat#ab154851, RRID:AB_2904567), anti-PRKAR2A (ProteinTech Cat# 10142-2-AP), anti-phospholamban (Cell Signaling Technology Cat# 14562, RRID:AB_2798511), anti-phospho-phospholamban -pS16/T17 (Cell Signaling Technology Cat# 8496, RRID:AB_10949102), anti-Ryanodine receptor 2 (Abcam Cat#ab196355, RRID:AB_2904568), anti-p-Ryanodine receptor 2-pS2808 (Abcam Cat# ab59225, RRID:AB_946327), anti-TnI (Cell Signaling Cat# 4002), anti-TnI-pS23/S24 (Cell Signaling Cat# 4004), anti-V5 (Sigma-Aldrich Cat# V8137, RRID:AB_261889), anti-PKA Catalytic subunit (Abcam Cat# ab26322, RRID:AB_2170049), HRP-conjugated anti-mouse (Cell Signaling Technology Cat# 7076, RRID:AB_330924) and HRP-conjugated anti-rabbit (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) were used for detection.

Techniques: Recombinant, SYBR Green Assay, In Vitro, Activity Assay, Viability Assay, Sequencing, esiRNA, CRISPR, Knock-Out, Plasmid Preparation, Software

Journal: Journal of molecular and cellular cardiology

Article Title: Partial and complete loss of myosin binding protein H-Like cause cardiac conduction defects

doi: 10.1016/j.yjmcc.2022.04.012

Figure Lengend Snippet: Line scanning confocal images from isolated atrial cardiomyocytes loaded with Cal520 calcium indicator dye and the associated calcium transient profiles from WT (A) and Null (B) groups. Single 1000 ms transient shown with standard deviation across a single cell. (C) Peak calcium transient amplitude at 1000 ms and 500 ms pacing cycle lengths (left) and the heterogeneity index (right) of peak amplitude measured along the length of each cell. N = 3 mice per group, two male, one female WT mice and one male, two female Null mice with averages of 8, 8, and 13 WT cells; 23, 13, and 13 Null cells. (D) Time to peak calcium release at each cycle length (left) and heterogeneity index (right) of time to peak calcium release. (E) The maximal rate of calcium release (+dR/dt) at each frequency and the heterogeneity index of the maximal rate of calcium release. (F) Structured illumination microscopy images of isolated atrial cardiomyocytes stained with anti-ryanodine receptor 2 antibodies and fluorescent conjugated phalloidin to mark actin. (G) Quantification of RyR2 puncta per area, the percent occurrence of cells exhibiting longitudinal lines of RyR2 puncta between myofibers, and the percent of cardiomyocytes with RyR2 puncta outside the peri-Z-disk region. N = Average of all cells from WT, 10; Het 5, Null 5 animals. WT 18 cells, Het 12 cells, Null 11 cells. (H) Otsu thresholding of structured illumination microscopy images and quantification of average RyR2 cluster size, as well as the cluster size of each cell at the 25th percentile and median values. Clusters measured from N = WT 32, Het 8, Null 17 individual cardiomyocytes. (I) Histogram of RyR2 cluster size from thresholded images. N = WT 32, Het 8, Null 17 Individual cardiomyocytes from 2 male, 1 female WT mouse, two female Het mice, and one male, one female null mouse.

Article Snippet: Primary antibodies were used against MyBP-HL (Pocono, custom), epitope and antibody validation in , cMyBP-C (Santa Cruz E7), contactin-2 (R&D Systems AF4439) [ 21 ], ryanodine receptor 2 (AbCam GR3250452–2).

Techniques: Isolation, Standard Deviation, Microscopy, Staining

Journal: bioRxiv

Article Title: STK25 inhibits PKA signaling by phosphorylating PRKAR1A

doi: 10.1101/2022.01.27.478084

Figure Lengend Snippet: A) Immunoblot of PRKAR1A phosphorylation sites S77 and S83 in STK25 +/+ and STK25 -/- cardiomyocyte protein lysates. B) Immunoblots of phosphorylation sites S77 and S83 of PRKAR1A in STK25 -/- cardiomyocytes transfected with empty vector (EV), wild type STK25 and kinase dead K49R/T174A (loaded in duplicate). C) Forskolin (10μM) stimulated STK25 +/+ and STK25 -/- cardiomyocytes immunoblotted for phosphorylation of PRKAR1A and RYR2. D) Immunoprecipitation of Flag-STK25 expressed in HEK293T cells and immunoblotted for PRKAR1A and GM130 (positive control binding partner). E) Co-immunoprecipitation of PRKAR1A-V5 with STK25 and PRKACA in HEK293T cells treated with forskolin (10μM). F) In vitro kinase assay of purified STK25 and PRKAR1A, immunoblotted for phosphorylation of S83 of PRKAR1A.

Article Snippet: For Western blotting antibodies include: HRP conjugated anti-GAPDH (Cell Signaling Technology Cat# 3683, RRID:AB_1642205), anti-STK25 (Abcam Cat# ab157188, RRID:AB_2725788), anti-Flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-GM130 (Cell Signaling Technology Cat# 12480, RRID:AB_2797933), anti-PRKAR1A (Abcam Cat# ab139695, RRID:AB_2893184), anti-pS77 PRKAR1A (Abcam Cat#ab139682, RRID:AB_2904566), anti-pS83 PRKAR1A (Abcam Cat#ab154851, RRID:AB_2904567), anti-phospholamban (Cell Signaling Technology Cat# 14562, RRID:AB_2798511), anti-phospho-phospholamban -S16/T17 (Cell Signaling Technology Cat# 8496, RRID:AB_10949102), anti-Ryanodine receptor 2 (Abcam Cat#ab196355, RRID:AB_2904568), anti-p-Ryanodine receptor 2-S2808 (Abcam Cat# ab59225, RRID:AB_946327), anti-V5 (Sigma-Aldrich Cat# V8137, RRID:AB_261889), anti-PKA Catalytic subunit (Abcam Cat# ab26322, RRID:AB_2170049), HRP-conjugated anti-mouse (Cell Signaling Technology Cat# 7076, RRID:AB_330924) and HRP-conjugated anti-rabbit (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) were used for detection.

Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Binding Assay, In Vitro, Kinase Assay, Purification