Journal: Frontiers in Cardiovascular Medicine

Article Title: Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression

doi: 10.3389/fcvm.2022.763544

Figure Lengend Snippet: CCN5 prevents cardiac fibrosis in mdx/utrn (±) mice. (A) Experimental scheme for panels (B–E) . mdx/utrn (±) mice were injected with AAV.9-Con or CCN5 into the tail vein, and hearts were harvested for experiments 8 weeks later. Age-matched WT mice are shown in comparison. (B) Hearts were sectioned and stained with trichrome. Blue areas indicate fibrotic tissue and red areas indicate normal tissue. (C) The ratio of fibrotic area over total tissue of the stained hearts was plotted. (D) Proteins obtained from cardiac tissue were immunoblotted with antibodies against CCN5, α-SMA, collagen I, SERCA2a, RyR2, NCX1, phosphorylated phospholamban (p-PLN), t-PLN and α-tubulin. (E) Protein bands on western blots were scanned and plotted. n = 6. * p < 0.05 and ** p < 0.01.

Article Snippet: Transferred blots were blocked with 5% non-fat skim milk and incubated with antibodies against mouse monoclonal CCN5 (1:1,000, Sigma, #WH0008839M9), mouse monoclonal α-SMA (1:1,000, Sigma-Aldrich, #A5228), goat polyclonal collagen I (1:1,000, Abcam, ab34710), rabbit polyclonal SERCA2a (1:1,000, a custom antibody from 21st Century Biochemicals), rabbit polyclonal RyR2 (1:1,000, Alomone Lab, ARR-002), rabbit monoclonal NCX1 (1:1,000, Abcam, EPR12739), rabbit polyclonal p-PLN (1:1,000, Badrilla, A010-12AP), mouse monoclonal t-PLN (1:1,000, Badrilla, A010-14), and mouse monoclonal α-tubulin (1:3,000, Santa Cruz, #sc-8035) for 12–16 h at 4°C.

Techniques: Injection, Staining, Western Blot

Journal: The Journal of Poultry Science

Article Title: Egg Development After In Vitro Insemination in Japanese Quail ( Coturnix japonica )

doi: 10.2141/jpsa.2023001

Figure Lengend Snippet: Presence of CCNB1, c-MOS, ITPR1, and RYR3 proteins in quail eggs and their down-regulated expression after ICSI. (A) Detection of CCNB1, c-MOS, ITPR1, and RYR3 proteins in ovulated quail eggs. (B) Western blot analysis 3 h after ICSI with injection of PLCZ1, CS, and ACO2 cRNA or the solvent alone. (C) Western blot analysis 3 h after ICSI with PLCZ1 cRNA alone, CS and ACO2 cRNAs, or the solvent alone. (D) Quantification of immunoreactivity after ICSI. Band intensities were quantified and expressed as the means ± standard deviations of three samples. Values with different letters are significantly different (P < 0.01).

Article Snippet: Rabbit anti-rat ITPR1 antibody (Alomone Labs Ltd., Jerusalem, Israel) and mouse anti-chicken RYR antibody (GeneTex, Inc., Irvine, CA, USA) were used to detect ITPR1 and RYR3, respectively.

Techniques: Expressing, Western Blot, Injection

Journal: The Journal of Poultry Science

Article Title: Egg Development After In Vitro Insemination in Japanese Quail ( Coturnix japonica )

doi: 10.2141/jpsa.2023001

Figure Lengend Snippet: Changes in ITPR1, RYR3, CCNB1, and c-MOS protein expression levels in quail eggs following in vitro insemination with different sperm numbers. (A–C) Western blot analysis 3 h after in vitro insemination with 2 × 10 2 (A), 2 × 10 3 (B), or 2 × 10 4 (C) sperm, respectively. Numbers 1–6 represent each of the six eggs treated with sperm. Egg extracts after ICSI or PBS treatment were used as the control. (D) Quantification of immunoreactivity after in vitro insemination. Band intensities were quantified and are expressed as means ± standard deviations. The sample number of PBS- and ICSI-treated eggs is three each. Band intensities in these three samples were measured in each membrane.

Article Snippet: Rabbit anti-rat ITPR1 antibody (Alomone Labs Ltd., Jerusalem, Israel) and mouse anti-chicken RYR antibody (GeneTex, Inc., Irvine, CA, USA) were used to detect ITPR1 and RYR3, respectively.

Techniques: Expressing, In Vitro, Western Blot

Journal: International Journal of Cardiology. Heart & Vasculature

Article Title: Anti-inflammatory effects of endothelin receptor blockade in left atrial tissue of spontaneously hypertensive rats

doi: 10.1016/j.ijcha.2022.101088

Figure Lengend Snippet: Effects of macitentan on the calcium handling in LA in SHR. (a) Representative immunoblots showing expression of α1C, NCX1, CSQ and RyR2 from SHR treated with MAC or DOX versus SHR-CTL (b-g) Quantification of immunoblots (N = 6–9 per group) (h) Representative immunoblots showing expression of SERCA2a, CaMKIIδ and PLB. Actin controls for pS16, pT17 and pS10 are not shown (i-m) Quantification of immunoblots (N = 6–9); # P < 0.05. Note that some blots/bands of housekeeping proteins used for normalization are identical (e.g. in (a) GAPDH blot/bands shown below CSQ and GAPDH blot/bands shown below RyR) as they are derived from the same membrane.

Article Snippet: Quality of transfer was probed using Ponceau S. Primary antibodies used for detection of protein expression and phosphorylation were 1) on 4–20% gradient gels (BioRad, Germany): anti-CaMKII (50 kD, Badrilla, A010-55AP, 1:5,000), anti-CSQ (55 kD, Thermo Scientific, PA1-913, 1:2,500), anti-GAPDH (34 kD, Calbiochem, CB1001, 1:50,000), anti-IP 3 R2 (313 kD, Abcam, ab77838, 1:1,000), anti-NCX1 (120 kD, Thermo Scientific, MA1-4672, 1:1,000), anti-SERCA2a (100 kD, Badrilla, A010-20, 1:5,000), anti-RyR (565 kD, Thermo Scientific, MA3-916, 1:5,000), anti-pSer2808-RyR2 (Badrilla, A010-30, 1:5,000); 2) on 8% glycine gels: anti-Cav1.2a, i.e. cardiac type α1C subunit of the L-type Ca channel (250 kD, Alomone Labs, ACC-013, 1:200); and 3) on 14% tricine gels: anti-actin (44 kD, clone C4, MP Biomedicals #69100, 1:50,000), anti-PLB (25 kD, Badrilla, A010-14, 1:5,000), anti-Ser10-PLB (Badrilla, A010-10AP, 1:1,000), anti-pSer16-PLB (Badrilla, A010-12, 1:5,000), anti-pThr17-PLB (Badrilla, A010-13, 1:5,000).

Techniques: Western Blot, Expressing, Derivative Assay

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet: Discrete cAMP pools in adult mouse SAN cells (A) Diagrams highlighting localization and schematic representation of the Epac1-camps-based FRET biosensors (ICU3) in the cytosol (cyt; 1), plasma membrane (PM; 2), sarcoplasmic reticulum (SR; 3), myofilaments (MF; 4), and nucleus (nuc; 5). The ICU3 is linked to a Kras-derived sequence for PM localization, to a phospholamban (PLB)-derived sequence for SR localization, to a troponin T (TnT) for MF localization, and to a nuclear localization signal (NLS) sequence for nucleus localization. Exemplary super resolution images of adult wild-type mouse SAN cells expressing the indicated ICU3 biosensor in the cytosol (B), PM (C), SR (D), MF (E), and nucleus (F). The biosensor-associated fluorescence (YFP) is in magenta. Cells were immunostained with specific markers (in cyan) for the PM (caveolin 3), SR (ryanodine receptor 2 [RyR2]), MF (phalloidin; phal), and nucleus (DAPI). Merged images and corresponding line profile analysis (for dotted line) show high degree of overlap between the YFP fluorescence linked to the biosensor and the corresponding cellular marker in all cases, except in cells expressing the cytosolic sensor, as expected. Dotted squares highlight expanded regions in the solid squares. (G) Scatterplot of Pearson's correlation coefficient for cyt/cav3, PM/cav3, SR/RYR2, MF/phal, and nuc/DAPI (n > 8 SAN cells per condition). Kruskal-Wallis with Dunn's multiple comparisons test was used to test statistical differences in Pearson's correlation coefficient between non-target and targeted sensors. Scatterplot of the FRET ratio change in response to 10 μM forskolin (fsk) + 100 μM IBMX (H) and cAMP concentration-response curves (I) generated in HEK cells expressing the different ICU3 sensors (n > 5 cells per condition). For the cAMP concentration-response curves, cells expressing the different ICU3 sensors were exposed to increasing concentrations of the membrane-permeable cAMP analog 8CPT-cAMP. Kruskal-Wallis with Dunn's multiple comparisons test was used to compare fsk + IBMX responses, and the extra sum-of-squares F test was used to compare the cAMP EC 50 response between sensors. (J) Average FRET ratio traces (mean, solid lines; SEM, shadow) in response to 100 nM isoproterenol (iso) or 10 μM fsk from adult wild-type mouse SAN cells expressing the cytosolic, PM, SR, MF, or nuclear ICU3 biosensors (n > 5 cells from three preparations per condition). Scatterplots of ΔR/R 0 (K) and normalized (L) FRET responses after application of iso or fsk. Statistical differences were assessed with two-tailed Mann-Whitney test for comparisons between iso and fsk responses in (H) Statistical differences in fsk responses between the different biosensors in H were assessed with a Kruskal-Wallis with Dunn's multiple comparisons test. Statistical differences in normalized iso responses between the different groups were assessed using a one-way ANOVA with Tukey's multiple comparisons test. Significance (∗) was considered at P < 0.05. Exact p values are available in . Data represent mean ± SEM.

Article Snippet: For experiments in , cells infected with the different ICU3 biosensors were incubated with an anti-caveolin 3 antibody (1:200, Thermo-Fisher Scientific PA1-066) to label the PM, an anti-ryanodine receptor 2 (RyR2) antibody (1:200, Alomone Labs ARR-002) to label the SR, an Alexa Fluor 647 Phalloidin (1.3 U; Thermo-Fisher Scientific A22287) to label MF, or DAPI from the ProLong Diamond Antifade Mountant (Thermo-Fisher Scientific) to label the nucleus.

Techniques: Derivative Assay, Sequencing, Expressing, Fluorescence, Marker, Concentration Assay, Generated, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Deciphering cellular signals in adult mouse sinoatrial node cells

doi: 10.1016/j.isci.2021.103693

Figure Lengend Snippet:

Article Snippet: For experiments in , cells infected with the different ICU3 biosensors were incubated with an anti-caveolin 3 antibody (1:200, Thermo-Fisher Scientific PA1-066) to label the PM, an anti-ryanodine receptor 2 (RyR2) antibody (1:200, Alomone Labs ARR-002) to label the SR, an Alexa Fluor 647 Phalloidin (1.3 U; Thermo-Fisher Scientific A22287) to label MF, or DAPI from the ProLong Diamond Antifade Mountant (Thermo-Fisher Scientific) to label the nucleus.

Techniques: Recombinant, Software

Journal: Circulation. Arrhythmia and electrophysiology

Article Title: Aging Disrupts Normal Time-of-day Variation in Cardiac Electrophysiology

doi: 10.1161/CIRCEP.119.008093

Figure Lengend Snippet: Ca2+ handling protein expression or phosphorylation at ZT4 and ZT14 in adult hearts. A-B. Phosphorylation of PLB at serine16 was essentially non-detectable following 5min perfusion (A) and 1hr perfusion (to match optical mapping data collection timepoint, B). Band labeled ‘Iso’ in (B) was a single heart perfused with isoproterenol for a positive control for pPLB. C-D. PLB phosphorylation was higher at the Thr17 site at ZT4 vs. ZT14 following 5min perfusion (C). However, phosphorylation levels were reduced and were not different between ZT4 and ZT14 following 1hr of perfusion (D). Band labeled ‘Iso’ in (D) was a single heart perfused with isoproterenol for a positive control for pPLB. E-G. Total PLB (E), SERCA2a (F), and RyR2 (G) expression. α-tubulin was used as loading control. ZT4 n=4; ZT14 n=4; except RyR (G) where ZT4 n=3; ZT14 n=3. *p<0.05.

Article Snippet: 25 Anti-SERCA (1:5000, A010–20, Badrilla), RyR2 (1:2500, ARR002, Alomone), PLB (1:5000, A010–14, Badrilla), PLB-Thr17 (1:5000, A010–13, Badrilla), PLB-Ser16 (1:5000, A010–12, Badrilla), and α-tubulin (1:1000, ab52866, Abcam) were used as primary antibodies.

Techniques: Expressing, Labeling, Positive Control

Journal: Frontiers in Pharmacology

Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

doi: 10.3389/fphar.2021.727526

Figure Lengend Snippet: Flow cytometry detects RYR1 in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).

Article Snippet: The membrane was first incubated in tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk to reduce non-specific antibody binding, then incubated overnight at 4°C with primary antibody RYR1 (1:1,000, Alomone Labs, Jerusalem, Israel) prepared in TBS containing 0.05% Tween-20 and 5% non-fat dry milk.

Techniques: Flow Cytometry, Isolation, Staining, Negative Control, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

doi: 10.3389/fphar.2021.727526

Figure Lengend Snippet: RYR1 antibody selectivity. (A) Western blot shows positive detection of RYR1 (∼565 kD) in rat skeletal muscle lysate and no detection in rat heart or brain lysate. (B,C) Contour plots of RYR1 antibody testing. (B) Red population represents positive detection of RYR1 subtype in male LMCs. Grey population represents the LMC suspension containing no primary antibody. (C) No detection in male LMCs after co-incubation with competing peptide (CP; 1:50 dilution) for the RYR1 antibody. Data representative of three isolations for each sex ( n = 6).

Article Snippet: The membrane was first incubated in tris-buffered saline (TBS) containing 0.05% Tween-20 and 5% non-fat dry milk to reduce non-specific antibody binding, then incubated overnight at 4°C with primary antibody RYR1 (1:1,000, Alomone Labs, Jerusalem, Israel) prepared in TBS containing 0.05% Tween-20 and 5% non-fat dry milk.

Techniques: Western Blot, Incubation

Journal: Frontiers in Pharmacology

Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

doi: 10.3389/fphar.2021.727526

Figure Lengend Snippet: Flow cytometry detects RYR1 in freshly isolated LMCs. Scatter plots of LMC suspensions plotted as side scatter (SSC-W) vs. fluorescent signals. Black horizontal lines denote the gate set. Fluorescent signal appearing above gate indicates positive staining. (A) Negative control (Neg) showing background FITC fluorescence of cell population with no antibody staining. Another LMC suspension was used to identify (B, left ) the LMC population stained by α-SMA-FITC ( red box ) and (B, right ) the gate set for non-specific staining of Alexa-Fluor 647 conjugated secondary antibody without primary antibodies within the α-SMA-FITC gated region. (C) Positive detection of RYR1 above the Alexa-Fluor 674 gate in LMCs isolated from rat mesenteric LVs. Data representative of five isolations each from male and female rats ( n = 10).

Article Snippet: Then cells were divided into groups for incubation with and without primary antibody targeting the RYR1 subtype (Alomone Labs, Jerusalem, Israel; ARR-001) at a dilution of 1:200 and rotated at 4°C overnight.

Techniques: Flow Cytometry, Isolation, Staining, Negative Control, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation

doi: 10.3389/fphar.2021.727526

Figure Lengend Snippet: RYR1 antibody selectivity. (A) Western blot shows positive detection of RYR1 (∼565 kD) in rat skeletal muscle lysate and no detection in rat heart or brain lysate. (B,C) Contour plots of RYR1 antibody testing. (B) Red population represents positive detection of RYR1 subtype in male LMCs. Grey population represents the LMC suspension containing no primary antibody. (C) No detection in male LMCs after co-incubation with competing peptide (CP; 1:50 dilution) for the RYR1 antibody. Data representative of three isolations for each sex ( n = 6).

Article Snippet: Then cells were divided into groups for incubation with and without primary antibody targeting the RYR1 subtype (Alomone Labs, Jerusalem, Israel; ARR-001) at a dilution of 1:200 and rotated at 4°C overnight.

Techniques: Western Blot, Incubation