rabbit anti ryr2  (Alomone Labs)

Journal: Frontiers in Cardiovascular Medicine

Article Title: The inotropic and arrhythmogenic effects of acutely increased late I Na are associated with elevated ROS but not oxidation of PKARIα

doi: 10.3389/fcvm.2024.1379930

Figure Lengend Snippet: Expression and phosphorylation level of Ca 2+ handling proteins.

Article Snippet: Following denaturation, the proteins were separated on 5% (RyR2, pSer2809 RyR2, pSer2814 RyR2), 8% (PKARIα dimer, PKARIα monomer, pThr287 CaMKII, and CaMKIIδ), or 12.5% (PLB, pSer16 PLB) SDS-polyacrylamide gels, transferred to a nitrocellulose membrane and incubated with the following primary antibodies: rabbit polyclonal anti-pThr287 CaMKII (1:1,000, PhosphoSolutions, Aurora, CO, USA), rabbit polyclonal anti-CaMKIIδ (1:10.000, Thermo Fisher Scientific Inc. of Waltham, MA, USA), mouse monoclonal anti-PKARIα (1:1,000, BD Biosciences, Heidelberg, Germany), rabbit polyclonal anti-RyR2 antibody (1:10.000, Sigma-Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-pSer2809 RyR2 antibody (1: 1,000, Badrilla, Leeds, United Kingdom), rabbit polyclonal anti-pSer2814 RyR2 antibody (1:1,000, Badrilla, Leeds, United Kingdom), mouse monoclonal anti-PLB (1:10.000, Thermo Fisher Scientific Inc. of Waltham, MA, USA), rabbit polyclonal anti-pSer16 PLB (1:500, Badrilla, Leeds, United Kingdom), and mouse monoclonal anti-GAPDH (1:10.000, Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight.

Techniques: Expressing

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Immunofluorescence colocalization of BBLN with CAMK2D on a heart specimen of an 8-month-old, male Tg- BBLN mouse (scale bar, 40 μm). The immunofluorescence colocalization was performed with heart specimens of five different Tg- BBLN mice (Tg-1) and five different nontransgenic FVB controls. All immunofluorescence images are shown in Supplementary Fig. . b , Quantitative IB determination of cardiac contents of activated phospho-T287–CAMK2D, inactive phospho-T307–CAMK2D, total CAMK2D and BBLN in 8-month-old, Tg- BBLN mice (Tg-1) and age- and sex-matched nontransgenic FVB control mice. The lower control IB detects ATP5A1. Left: IB images and right: quantitative IB data (mean ± s.d., n = 9 mice per group, 5 males and 4 females; unpaired, two-tailed t -test; d.f. 16, t = 32.92, 8.467, 2.602 and 56.01; P = 3.9625 × 10 −16 , 2.63699 × 10 −7 , 0.01927 and 8.64361 × 10 −20 ). c , d , In vitro data show that recombinant BBLN protein enhanced the autophosphorylation of recombinant CAMK2D (200 nM) and the CAMK2D-mediated substrate phosphorylation of recombinant PDC and BBLN. Representative autoradiography images ( c ) and quantitative data ( d ) of BBLN-enhanced PDC phosphorylation by CAMK2D (50 nM) in vitro, in the presence and absence (w/o) of Ca 2+ +calmodulin (CALM) (mean ± s.d., n = 3 biological replicates, one-way ANOVA and Dunnett’s test; F (9,20) = 38.33; P = 0.9998, 0.3728, <0.0001 and <0.0001 BBLN+Ca 2+ +CALM versus Cont.+Ca 2+ +CALM; ** P = 0.0052 versus Cont.+Ca 2+ +CALM; * P = 0.0182, 0.0321, 0.0113 and 0.0135 versus Cont.+Ca 2+ +CALM). e , Quantitative IB determination of cardiac contents of phospho-T287–CAMK2D, total CAMK2D, phospho-S2813–RYR2 and BBLN (and BBLN–SxxA) in 8-month-old, male Tg- BBLN mice (Tg-2), nontransgenic FVB mice and Tg- BBLN mice (Tg-2) after 4 weeks of lentiviral transduction of miCamk2d (Tg- BBLN +miCamk2d) and Tg- BBLN –SxxA mice. The control IB detects ATP5A1. Quantitative data (left) and IB images (right) (mean ± s.d.; n = 4 male, 8-month-old mice per group, one-way ANOVA and Tukey’s test; F (3,12) = 111.4, 27.29, 62.58 and 52.25; upper left: P = 1.914 × 10 −7 , 5.873 × 10 −9 and 3.424 × 10 −8 ; lower left: P = 0.4303, 0.00001428 and 0.5147; upper right: P = 0.000007374, 8.287 × 10 −8 and 0.000004093; lower right: P = 0.0001016, 0.0933 and 0.001294); f , The left ventricular EF and the LVIDd of 8-month-old, male mice were determined by echocardiography (mean ± s.d., n = 6 mice per group, one-way ANOVA and Tukey’s test; F (3,20) = 54.28 and 5.562; P = 1.19 × 10 −9 , 3.58 × 10 −8 , 6.628 × 10 −8 EF; P = 0.004012, 0.1793 and 0.0449 LVIDd).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Immunofluorescence, Two Tailed Test, In Vitro, Recombinant, Autoradiography, Transduction

Journal: Nature Cardiovascular Research

Article Title: BBLN triggers CAMK2D pathology in mice under cardiac pressure overload and potentially in unrepaired hearts with tetralogy of Fallot

doi: 10.1038/s44161-023-00351-6

Figure Lengend Snippet: a , Immunoblot analysis detected reduced total cardiac RYR2 protein contents and increased CAMK2D-mediated phosphorylation of RYR2 on serine-2813 in Tg- BBLN mice (Tg-1) compared to non-transgenic FVB controls. Representative immunoblots (left panels), and quantitative immunoblot data (right panels) are shown. b , Cardiac ATP2A2 (SERCA2) levels are decreased in Tg- BBLN (Tg-1) hearts. The lower control blot detects ATP5A1. Representative immunoblots (left panels), and quantitative data (right panels) are shown. Data ( a , b ) are mean ± s.d. (n = 9 mice per group, 5 male, 4 female; age: 8 months). P-values were determined by the unpaired, two-tailed t-test (df=16; a , t = 9.4818, 4.9131; p = 5.7293e-08, p = 0.000156; b , t = 11.09, 0.3828; p = 6.42141e-09, p = 0.7069). c , Transcript levels of Ryr2 (left panel) and Atp2a2 (right panel) were determined by NGS in heart specimens of Tg- BBLN mice (Tg-1) and non-transgenic FVB mice. Data are mean ± s.d. (n = 4 male mice per group; age: 8 months). P-values were determined by the unpaired, two-tailed t-test (df=6; t = 7.4612, p = 0.000299 ( Ryr2 ); t = 33.28, p = 4.8989e-08 ( Atp2a2 )). d , BBLN transcript levels in right ventricular heart specimens of TOF patients with cyanosis were determined by microarray analysis (Affymetrix probe set ID: 204480_s_at) and negatively correlated with RYR2 (214044_at) and ATP2A2 (212361_s_at). Linear regression analysis was performed, and the Pearson correlation coefficient (r) and p-values (left panel, two-tailed, p = 0.0412; right panel, one-tailed, p = 0.0486) were determined (n = 11 TOF patients with cyanosis, 7 females, 4 males; age: 23.2 ± 5 months).

Article Snippet: The following antibodies were used for IB detection, immunohistochemistry and immunofluorescence: rabbit monoclonal anti-ATP2A2/SERCA2 antibody (9580; D51B11; Cell Signaling Technology); rabbit polyclonal anti-C9orf16 (anti-BBLN) antibodies (HPA020725; Prestige Antibodies; Sigma Life Sciences); rabbit polyclonal anti-CAMK2D antibodies (H00000817-DO1P; Abnova); mouse monoclonal anti-CAMK2D antibody, clone 1A8 (WH0000817M2; Sigma-Aldrich); rabbit monoclonal anti-CAMK2D antibody [EPR13095] (ab181052; abcam); rabbit polyclonal anti-phospho-Thr287 CAMKII (beta, gamma, delta) antibodies (PA5-37833; Invitrogen by ThermoFisher Scientific); rabbit monoclonal anti-phospho-Thr286/287 CAMK2 (alpha, beta, gamma, delta) antibody (D21E4; 12716; Cell Signaling Technology); rabbit polyclonal anti-phospho-Thr305 CAMK2 (alpha, beta, gamma, delta) antibodies (Thr307 in mouse CAMK2D) (Abnova PAB29254; B1SA01040G00470); rabbit monoclonal anti-Desmin antibody [Y66] (ab32362, abcam); mouse monoclonal anti-ATP5A antibody [15H4C4] (ab14748; abcam); rabbit monoclonal anti-MLKL antibody (D6W1K) (mouse specific; 37705; Cell Signaling Technology); rabbit monoclonal anti-phospho-S345–MLKL antibody [EPR9515 (ref. )] (ab 196436; abcam); rat monoclonal anti-MLKL antibody [3H1] (ab243142; abcam); mouse monoclonal anti-phospho-S345-MLKL antibody (MABC1158, Clone 7C6.1; EMD Millipore Corporation); rabbit monoclonal anti-phospho-S358-MLKL (D6H3V) antibody (mAb No. 91689; Cell Signaling Technology); rabbit monoclonal anti-MLKL (D2I6N) antibody (mAb No. 14993 Cell Signaling Technology); rabbit polyclonal anti-RYR2 antibodies (Invitrogen PA5-77717; ThermoFisher Scientific); rabbit polyclonal anti-phospho-Ser2814 RYR2 antibodies (CABP0624; AssayGenie); mouse monoclonal anti-α-Tubulin antibody, clone DM1A (T6199; Sigma); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG Fcγ Fragment Specific (115-036-071; Jackson ImmunoResearch Laboratories); peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Rabbit IgG, Fc Fragment-Specific (111-036-046; Jackson ImmunoResearch Laboratories); Protein A, Peroxidase Conjugate (539253-1MG; EMD Millipore Corporation); goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 ( A11034 ; Invitrogen by ThermoFisher Scientific); goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 ( A11004 ; Invitrogen by ThermoFisher Scientific).

Techniques: Western Blot, Transgenic Assay, Two Tailed Test, Microarray, One-tailed Test