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: 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: 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

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: Microcirculation (New York, N.Y. : 1994)

Article Title: Aging alters spontaneous and neurotransmitter-mediated Ca 2+ signaling in smooth muscle cells of mouse mesenteric arteries

doi: 10.1111/micc.12607

Figure Lengend Snippet: Transcript levels for ryanodine receptor isoforms in mesenteric artery smooth muscle cells from young and old mice. Data are from 22 with permission.

Article Snippet: Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003).

Techniques:

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Aging alters spontaneous and neurotransmitter-mediated Ca 2+ signaling in smooth muscle cells of mouse mesenteric arteries

doi: 10.1111/micc.12607

Figure Lengend Snippet: Representative immunofluorescence images depicting green fluorescence for RyR1 (top rows), RyR2 (center rows) and RyR3 (bottom rows) in 3 separate SMCs from MAs of (A) young and (B) old mice, (C) SMCs incubated with respective blocking peptides, and (D) SMC with primary omitted. ToPro nuclear stain (blue) is included in all images. Scale bars = 20 μm and apply to all panels.

Article Snippet: Thus prepared, slides were incubated 60 min in primary antibody [Alamone Labs, 1:250] for RyR1 (Cat. #ARR-001), RyR2 (Cat. #ARR-002), or RyR3 (Cat. #ARR-003).

Techniques: Immunofluorescence, Fluorescence, Incubation, Blocking Assay, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Ginkgolide B Maintains Calcium Homeostasis in Hypoxic Hippocampal Neurons by Inhibiting Calcium Influx and Intracellular Calcium Release

doi: 10.3389/fncel.2020.627846

Figure Lengend Snippet: GB reduced the expressions of proteins involved in intracellular calcium homeostasis in neurons exposed to hypoxia. (A) Representative western blots showing the expressions of Ca v 1.2, STIM1 and RyR2 proteins in hippocampal tissue from the various groups. (B) Quantification of the western blot data. (C) qPCR analysis of the mRNA levels of Ca v 1.2, STIM1 and RyR2. Datafor (B) and (C) are expressed as the mean ± SEM ( n = 3). ** P < 0.01 for hypoxia group vs. control group; # P < 0.05, ## P < 0.01 for hypoxia-GB group vs. hypoxia group (Student's t -test).

Article Snippet: The membranes were blocked for 1 h in non-fat milk and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-Ca v 1.2 (1:200, cat. #ACC-005, Alomone, Jerusalem, Israel), rabbit anti-stromal interaction molecule-1 (STIM1; 1:500, cat. #ACC-063, Alomone), rabbit anti-ryanodine receptor type-1 (RyR2; 1:200, cat. #ARR-001, Alomone) and rabbit anti-β-actin (1:2,000, cat. #4970, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats

doi: 10.3390/ijms21249593

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti-Ryanodine Receptor 1 , Ryanodine Receptor 1/ICC , Rabbit polyclonal , 1:200 , Alomone Labs.

Techniques: Marker