phospho ctni  (Bio-Rad)

Journal: bioRxiv

Article Title: Developmental Hypoxia Increases Susceptibility to Cardiac Ventricular Arrhythmias in Adult Offspring

doi: 10.64898/2026.01.22.701057

Figure Lengend Snippet: Data show the mean ± SD with each sample as dot plots for the protein abundance of phospholamban (PLN, B), phospho-PLN-T17 (C), ratio of phospho-PLN-T17 to PLN (D), cardiac troponin I (cTnI, E), phospho-cTnI (F), ratio of phospho-cTnI to cTnI (G), calsequestrin-1 (H), SERCA2 (I), ratio of SERCA2 to PLN (J), sodium-calcium exchanger 1 (NCX1, K), L-type calcium channel (LTCC, L), ryanodine receptor 2 (RYR2, M), phospho-RYR2-S2808 (N), ratio of phospho-RYR2-S2808 to RYR2 (O) and Connexin43 (Cx43, P). Data was analysed using an unpaired Student’s T-Test for all proteins except PLN, cTnI, NCX and phospho-cTnI/cTnI ratio which used a Mann-Whitney test (data not normally distributed). P<0.05 was considered significant. Normoxia = closed circles, Hypoxia = open circles.

Article Snippet: Membranes were incubated with primary antibodies (PLN [1/5000 dilution] – MA3-922, InVitrogen; phospho-PLN-T17 [1/1000 dilution] – AP0910, ABclonal; cTnI [1/1000 dilution] – MCA1208, Bio-Rad; phospho-cTnI [1/1000 dilution] – MCA2780, Bio-Rad; Calsequestrin-1 [1/5000 dilution] – PA1-913, InVitrogen; SERCA2 [1/2000 dilution] – ab3625, Abcam; NCX1 [1/1000 dilution] – R3F1, Swant; LTCC [1/1000 dilution] – ab2864, Abcam; RYR2 [1/1000 dilution] – ab302716, Abcam; phospho-RYR2-S2808 [1/1000 dilution] – PA5-105712, InVitrogen) overnight at 4°C, before secondary antibody incubation (IRDye® 800CW Goat anti-mouse for PLN, cTnI, phospho-cTnI, NCX1 and LTCC, IRDye® 800CW goat anti-rabbit for phospho-PLN-T17, Calsequestrin-1, SERCA2, RYR2 and phosphor-RYR2-S2808, both 1/20 000 dilution) at room temperature for one hour.

Techniques: Quantitative Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Desmoglein-2 deficiency results in cardiac dysfunction by compromising both Z-disc- and intercalated disc-mediated mechanotransduction

doi: 10.1101/2025.10.03.680335

Figure Lengend Snippet: Cropped images of western immunoblotting membranes incubated with anti-cMyBP-C, -cMyBP-C-P ser282 , -GAPDH, -cTnI, and -cTnI-P ser23/24 antibodies, and their respective immunoblot quantification. Tissue lysates were prepared from RV (a-b) and LV (c-d) free walls of adolescent and adult WT and Dsg2 mut/mut mice. Data are shown as mean ± SE. Statistical significance was assessed by unpaired Student t-test between adolescent Dsg2 mut/mut vs. WT mice; # P < 0.05 and ## P < 0.01. Number of biological replicates: WT n = 4 and Dsg2 mut/mut n = 5.

Article Snippet: Next, the membranes were placed in the iBind TM flex western device and probed with primary antibodies: rabbit polyclonal anti-GAPDH (Santa Cruz (F L-335): sc-25778) at 1:1000 dilution, rabbit polyclonal anti-MYBPC3-P ser282 (custom-made, gift from Dr. Sakthivel Sadayappan, University of Cincinnati) at 1:2000 dilution; mouse monoclonal anti-MYBPC3 (Santa Cruz (E-7): sc-137180) at 1:400 dilution; rabbit polyclonal anti-cTnI-P ser23/24 (Cell Signaling; Cat #4004) at 1:200 dilution; mouse monoclonal anti-cTnI total (TI-1, Developmental Studies Hybridoma Bank, University of Iowa) at 1:400 dilution, and rabbit polyclonal anti-α-Actinin 2 (GeneTex #GTX103219) at 1:500 dilution.

Techniques: Western Blot, Incubation

Journal: iScience

Article Title: High-throughput longitudinal electrophysiology screening of mature chamber-specific hiPSC-CMs using optical mapping

doi: 10.1016/j.isci.2023.107142

Figure Lengend Snippet: GCaMP6f repeated measures to determine effect of daily isoproterenol pulses on hiPSC-CM function (A) Daily measurement of GCaMP6f spontaneous calcium flux in hiPSC-CM monolayers confirmed pulsing of isoproterenol (0.2μM). At each time point, isoproterenol-treated monolayers’ spontaneous beat rate increased significantly (red symbols, n = 21; ∗∗∗∗p < 0.0001, unpaired t test compared to control, vehicle treated, n = 8, black symbols). (B) On day 5 of this protocol baseline recordings of spontaneous activity was recorded using the cardiac EP plate reader; these are representative traces from each group. Upstroke of the calcium transient is automatically determined and colored green while the calcium transient decay from peak is labeled red. (C) Isoproterenol pulsing increased spontaneous beat rate (Pulsed ISO = 0.65 ± 0.12Hz, n = 8 vs. Control (Vehicle) = 0.46 ± 0.09Hz, n = 16 monolayer technical replicates; ∗∗∗p = 0.0006, unpaired t test). (D) CaTD80 was shorter in ISO-pulsed monolayers (0.623 ± 0.11s, n = 16) relative to control (0.809 ± 0.02s, n = 8); ∗∗∗p = 0.0006, unpaired t test. (E) Calcium transient triangulation was significantly less in ISO-pulsed monolayers (0.309 ± 0.07s, n = 16) compared to control (0.467 ± 0.02s, n = 8) ∗∗∗∗p < 0.0001, unpaired t test. (F) Calcium transient amplitude was not different at baseline between the two groups. (G) Conduction velocity was greater in monolayers pulsed daily with ISO (26.4 ± 5.3 cm/s, n = 16 vs. 20.1 ± 4.9 cm/s, n = 8; ∗∗p = 0.009, unpaired t test). (H) Calcium transient upstroke slope (+dF/dt) was faster in ISO-pulsed monolayers. (I) Western blot analysis probing for SERCA2a expression (top) and phospho-cTnI (middle). Total protein stain (bottom) used for normalization and equal protein loading control. (J) SERCA2a protein expression on day 5 was greater in monolayers treated daily with ISO. ∗∗p = 0.001, unpaired t test, n = 4 per group. (K) In response to acute ISO on day 5, pulsed monolayers beat rate increased to a greater value than control (83.3 ± 13.8 beat per minute (BPM), n = 8 vs. 56.67 ± 20.0BPM, n = 4). (L) Conduction velocity increased to greater values following acute ISO treatment in monolayers with a history of ISO pulsing (48.9 ± 13.2 cm/s, n = 8 vs. 30.1 ± 8.4 cm/s, n = 4 technical replicates) ∗p = 0.02, unpaired t tests. Data are expressed as mean ± standard deviation.

Article Snippet: Primary antibodies used include mouse anti β-myosin heavy chain (A4.951, Developmental Studies Hybridoma Bank), mouse anti-MLC-2a (Synaptic Systems), mouse anti-GFP (Millipore Sigma), mouse anti-SERCA2a (Thermo Fisher, MA3-919), and rabbit anti phospho-cTnI (Cell Signaling Technology, 4004S).

Techniques: Control, Activity Assay, Labeling, Western Blot, Expressing, Staining, Standard Deviation

Journal: iScience

Article Title: High-throughput longitudinal electrophysiology screening of mature chamber-specific hiPSC-CMs using optical mapping

doi: 10.1016/j.isci.2023.107142

Figure Lengend Snippet:

Article Snippet: Primary antibodies used include mouse anti β-myosin heavy chain (A4.951, Developmental Studies Hybridoma Bank), mouse anti-MLC-2a (Synaptic Systems), mouse anti-GFP (Millipore Sigma), mouse anti-SERCA2a (Thermo Fisher, MA3-919), and rabbit anti phospho-cTnI (Cell Signaling Technology, 4004S).

Techniques: Virus, Plasmid Preparation, Recombinant, Membrane, Isolation, Software

Journal: iScience

Article Title: High-throughput longitudinal electrophysiology screening of mature chamber-specific hiPSC-CMs using optical mapping

doi: 10.1016/j.isci.2023.107142

Figure Lengend Snippet:

Article Snippet: Phospho-cTnI antibody , Cell Signaling Technology , Cat#4004S; RRID: AB_2940841.

Techniques: Virus, Plasmid Preparation, Recombinant, Membrane, Isolation, Software

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A

doi: 10.1016/j.jbc.2022.102767

Figure Lengend Snippet: Effects of λPP and PKA treatment on myofilament protein phosphorylation levels. A , pro-Q Diamond phospho-protein ( top ) and SYPRO-Ruby total protein staining ( bottom ) of cardiac myofibrils (CMFs) isolated from three different hearts before (PRE), after λPP, and after λPP/PKA-treatment. The position of cMyBP-C and cTnI are indicated by black and green arrows , respectively. B , cMyBP-C phosphorylation levels were determined by Western blot using phosphoserine 282–specific and total cMyBP-C antibodies. C , relative cMyBP-C phosphorylation levels determined by Pro-Q/SYPRO staining ( left ) and Western blot ( right ) of samples before treatment (PRE) and after λPP and λPP/PKA treatment. Bar plots are means ± SD. Comparisons with (PRE) were done using Brown-Forsythe and Welch ANOVA and Dunnett T3 test ( § p < 0.05).

Article Snippet: For immunoblotting using the polyclonal antibodies against phospho-cTnI (1:500 in TBS-T and 1% (w/v) bovine serum albumin [BSA], Cell Signaling, 4004) and total cTnI (1:1000 in TBS-T and 1% (w/v) semiskimmed milk powder, Cell Signaling, 4002), the membrane was blocked for 1 h in TBS-T and 5% (w/v) of the corresponding blocking reagent before overnight incubation with the primary antibody at 4 °C.

Techniques: Phospho-proteomics, Staining, Isolation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A

doi: 10.1016/j.jbc.2022.102767

Figure Lengend Snippet: Modulation of cardiac troponin I phosphorylation in ventricular trabecul a e by recombinant troponin exchange. A , example Phos-tag-Western blots against cTnI from experimental ventricular trabeculae, either untreated or λPP-treated, followed by exchange with either nonphosphorylated or bisphosphorylated recombinant rat troponin complex. Troponin I was detected with the mouse monoclonal antibody 84 against the central region (residues 118–127 in the rat cardiac sequence). B , densitometric analysis and plots of mol incorporated phosphate per mol cTnI (mol Pi/mol cTnI). C , densitometric analysis of cTnI phosphorylation levels from single trabeculae showing the relative percentages of cTnI species, summarized in ( D ). Bar plots are means ± SD. Statistics in ( B ) are Kruskal–Wallis test with Dunn’s multiple comparisons test against the λPP-treated cTn-2P group (ns not significant, §§ p < 0.01) and unpaired two-tailed t test for the untreated groups (∗∗∗∗ p < 0.0001).

Article Snippet: For immunoblotting using the polyclonal antibodies against phospho-cTnI (1:500 in TBS-T and 1% (w/v) bovine serum albumin [BSA], Cell Signaling, 4004) and total cTnI (1:1000 in TBS-T and 1% (w/v) semiskimmed milk powder, Cell Signaling, 4002), the membrane was blocked for 1 h in TBS-T and 5% (w/v) of the corresponding blocking reagent before overnight incubation with the primary antibody at 4 °C.

Techniques: Phospho-proteomics, Recombinant, Western Blot, Sequencing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A

doi: 10.1016/j.jbc.2022.102767

Figure Lengend Snippet: cTnI and cMyBP-C phosphorylation control cardiac length-dependent activation. Force-calcium titration of untreated ( A and B ) and λPP-treated trabeculae ( C and D ) exchanged with either nonphosphorylated ( A and C ) or bisphosphorylated cTn complex ( B and D ) at short (1.9 μm, open symbols) and long sarcomere length (2.3 μm, closed symbols). The dotted and dashed lines in ( B ) and ( D ) represent the Hill fits shown in ( A ) and ( C ), respectively. E , force-calcium relation of λPP-treated and subsequently PKA-treated trabeculae exchanged with bisphosphorylated cTn at short and long sarcomere length. F , summary plot showing the increase in calcium sensitivity (ΔpCa 50 ) upon sarcomere length increase for all experimental groups. Means ± SD, ( A ) n = 12 to 15, ( B ) n = 11 to 14, ( C ) n = 9, ( D ) n=8 to 10, and ( E ) n = 9. Statistical significance of differences was assessed with Brown-Forsythe and Welch ANOVA, followed by Dunnett’s T3 multiple comparisons test: ns not significant, § p < 0.05 and §§ p < 0.01.

Article Snippet: For immunoblotting using the polyclonal antibodies against phospho-cTnI (1:500 in TBS-T and 1% (w/v) bovine serum albumin [BSA], Cell Signaling, 4004) and total cTnI (1:1000 in TBS-T and 1% (w/v) semiskimmed milk powder, Cell Signaling, 4002), the membrane was blocked for 1 h in TBS-T and 5% (w/v) of the corresponding blocking reagent before overnight incubation with the primary antibody at 4 °C.

Techniques: Phospho-proteomics, Control, Activation Assay, Titration

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A

doi: 10.1016/j.jbc.2022.102767

Figure Lengend Snippet: Pearson correlation analysis of force pCa 50 and ΔpCa 50 versus the percentage Ser22/23 bisphosphorylated cTnI. A , B , and C ,: data from trabeculae with high levels of cMyBP-C phosphorylation. D and E , and F , data from λPP-treated trabeculae with low levels of cMyBP-C phosphorylation.

Article Snippet: For immunoblotting using the polyclonal antibodies against phospho-cTnI (1:500 in TBS-T and 1% (w/v) bovine serum albumin [BSA], Cell Signaling, 4004) and total cTnI (1:1000 in TBS-T and 1% (w/v) semiskimmed milk powder, Cell Signaling, 4002), the membrane was blocked for 1 h in TBS-T and 5% (w/v) of the corresponding blocking reagent before overnight incubation with the primary antibody at 4 °C.

Techniques: Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A

doi: 10.1016/j.jbc.2022.102767

Figure Lengend Snippet: cMyBP-C binds cTn in a cTnI-phosphorylation and calcium-dependent manner. A , microscale thermophoresis (MST) curves for nonphosphorylated C0C2 (C0C2-0P) and PKA trisphosphorylated C0C2 (C0C2-3P) binding to nonphosphorylated (cTn-0P) and cTnI bisphosphorylated cTn (cTn-2P) at low Ca 2+ concentrations (∼pCa 7.4). Left ΔF norm (%): the MST signal change is expressed as the percentage change of the normalized fluorescence (ΔF norm ), whereby F norm is defined as F 1 /F 0 (fluorescence values before F 0 and after F 1 infrared laser activation) and the baseline F norm (the mean F norm of the unbound target) is subtracted from all data points of the same curve. Right normalized binding: the fraction bound is plotted against ligand concentration, whereby all ΔF norm values of a curve are divided by the curve amplitude, resulting in normalized fraction bound. Inset bar plot of the dissociation constants ( K D ). B , same as in ( A ), except at high Ca 2+ concentrations (pCa 3.0). C , normalized MST-binding curves for C0C2 ( black ) and C0C2-3P ( purple ) titrated against Alexa647-labeled NcTnC. D , hypothetical model of the phosphorylation-dependent interaction of NcMyBP-C with cardiac troponin. Please see text for details. Means ± SD, n = 3 to 5. Statistical significance of differences was assessed with one-way ANOVA, followed by Tukey’s post hoc test ( A ) and Kruskal–Wallis test on ranks, followed by Dunn’s ( B ) § p < 0.05, §§ p < 0.01, §§§ p < 0.001. C , two-tailed unpaired t test ∗ p < 0.05.

Article Snippet: For immunoblotting using the polyclonal antibodies against phospho-cTnI (1:500 in TBS-T and 1% (w/v) bovine serum albumin [BSA], Cell Signaling, 4004) and total cTnI (1:1000 in TBS-T and 1% (w/v) semiskimmed milk powder, Cell Signaling, 4002), the membrane was blocked for 1 h in TBS-T and 5% (w/v) of the corresponding blocking reagent before overnight incubation with the primary antibody at 4 °C.

Techniques: Phospho-proteomics, Microscale Thermophoresis, Binding Assay, Fluorescence, Activation Assay, Concentration Assay, Labeling, Two Tailed Test