Journal: Communications Biology

Article Title: A maladaptive feedback mechanism between the extracellular matrix and cytoskeleton contributes to hypertrophic cardiomyopathy pathophysiology

doi: 10.1038/s42003-022-04278-9

Figure Lengend Snippet: Immunoblot analysis of L-type calcium channel ( a , b ) and β 1 integrin ( c , d ) protein expression performed on total heart homogenate pooled from groups of 5 pre- (10–15-wk-old) or post-cardiomyopathic (30–50-week-old) cTnI-G203S mice and age-matched wt counterparts. Representative immunoblots probed with L-type calcium channel α 1C subunit (Ca V 1.2, a ) or β 1 integrin ( c ) antibody, then GAPDH monoclonal antibody. Densitometry analysis of Ca V 1.2 ( b ) or β 1 integrin ( d ) protein expression, normalized to associated GAPDH expression. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( b ) or Kruskal-Wallis test ( d ) determined statistical significance. Densitometry analysis of relative mTOR expression (calculated as Phospho-mTOR/Total mTOR) performed on cytoplasmic ( e ) and nuclear ( f ) fractions pooled from groups of five pre- or post-cardiomyopathic cTnI-G203S mice and age-matched wt counterparts. β-tubulin and histone H2B antibodies were used as loading controls for cytoplasmic and nuclear fractions respectively. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( e ) or Kruskal-Wallis test ( f ) determined statistical significance. g Schematic indicating structural-functional link between the L-type calcium channel, cytoskeletal network, mitochondria, integrin and the extracellular matrix in wt and cTnI-G203S cardiac myocytes. A disrupted cytoskeletal architecture in cTnI-G203S cardiac myocytes may trigger a maladaptive feedback mechanism between increased cytoskeletal and extracellular matrix stiffness, resulting in a hypermetabolic mitochondrial state.

Article Snippet: Blots were probed with the following primary antibodies: rabbit polyclonal anti-Ca V 1.2 (Alomone, ACC-003, 1:200) or rabbit monoclonal anti-β 1 integrin (D6S1W) (Cell Signaling Technology 34971, 1:1000).

Techniques: Western Blot, Expressing, Functional Assay

Journal: Neural Regeneration Research

Article Title: L- and T-type Ca 2+ channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma

doi: 10.4103/1673-5374.360277

Figure Lengend Snippet: Changes in protein levels of L- and T-type Ca 2+ channel subunits in COH retinas. (A) Representative immunoblots showing the expressions of Ca V 1.2 (a L-type Ca 2+ channel subunit) and Ca V 3.1, Ca v 3.2, and Ca V 3.3 (T-type Ca 2+ channel subunits) in sham-operated (control) and COH retinal extracts at the indicated post-operational times. (B–E) Bar charts showing the densitometric quantification of immunoreactive bands of Ca v 1.2 (B), Ca v 3.1 (C), CaV3.2 (D), and Ca V 3.3 (E) under the conditions as shown in A. (F) Representative immunoblots showing the expression of Brn3a, a RGC marker, in control and COH retinal extracts at the indicated post-operational times. (G, H) Bar charts showing Brn3a protein levels (G) and Ca v 1.2/Brn3a ratios (H) under the conditions as shown in A and F. Data are presented as the mean ± SEM ( n = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control (one-way analysis of variance test with Bonferroni’s post hoc test (for B–D, G and H) and non-parametric Kruskal-Wallis test (for E)). COH: Chronic ocular hypertension; RGC: retinal ganglion cell.

Article Snippet: The primary antibodies used in this study were mouse anti-Ca V 1.2 monoclonal antibody (1:50, Abcam, Cambridge, MA, USA, Cat# ab84814, RRID: AB_1860052), rabbit anti-Ca V 3.1 polyclonal antibody (1:100, Alomone, Jerusalem, Israel, Cat# ACC-021, RRID: AB_2039779), rabbit anti-Ca V 3.2 polyclonal antibody (1:100, Alomone, Cat# ACC-025, RRID: AB_2039781), rabbit anti-Ca V 3.3 polyclonal antibody (1:100, Alomone, Cat# ACC-009, RRID: AB_2039783), goat anti-Brn-3a polyclonal antibody (1:300, Santa Cruz Biotechnology, Santa Cruz, TX, USA, Cat# sc-31984, RRID: AB_2167511), and mouse anti-β-actin (1:10,000, Sigma-Aldrich, Cat# A5316, RRID: AB_476743).

Techniques: Western Blot, Expressing, Marker

Journal: PLoS ONE

Article Title: Modulation of Calcium-Dependent Inactivation of L-Type Ca 2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

doi: 10.1371/journal.pone.0027474

Figure Lengend Snippet: ( A ) Representative current traces recorded under control conditions (upper left panel) and in the presence of the specific β 2 AR agonist salmeterol alone (10 µM; left middle panel) or in combination with PKI 14–22 amide (10 µM; lower left panel). The bar graph represents D inact under different recording conditions (as indicated). The mean value of five cells recorded under control conditions was taken for comparison with four cells recorded under 10 µM salmeterol and five cells recorded under 10 µM salmeterol plus PKI 14–22 amide. Data are presented as means ± SEM of several independent experiments. *** P <0.001. Significance of salmeterol plus PKI (n = 5) versus salmeterol alone (n = 4) was calculated by Student's t test. The degree of inactivation is given by the normalized current amplitude of the mean postpulse I/V at +10 mV. ( B ) Close co-expression of the main modulator of CDI, PKA (green) and Ca V 1.2 (red) in cultured neurons. Yellow dots represent places were these two proteins are in close proximity. Data shown are representative pictures from several independent immunostainings and preparations of neurons. In all cases, omission of primary antibodies resulted without signal (negative control). ( C ) Indicated brain regions were immunostained with antibodies specific for PKARIIβ and Ca V 1.2. Thalamic regions LGN and VB revealed very strong interaction patterns in merged pictures. Association of these proteins is still present in hippocampus but on lower level. DG (dentate gyrus), PoDG (polymorph layer of the dentate gyrus).

Article Snippet: After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-Ca V 1.2 (1∶200, Alomone Labs, Israel); mouse anti-cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIβ, 1∶500, BD Bioscience, USA); rabbit anti-β 2 -AR (H-73 1∶400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1∶1000, Sigma, Germany); goat anti-AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phosphatase 2A (PP2A, Acris, Germany).

Techniques: Expressing, Cell Culture, Negative Control

Journal: PLoS ONE

Article Title: Modulation of Calcium-Dependent Inactivation of L-Type Ca 2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

doi: 10.1371/journal.pone.0027474

Figure Lengend Snippet: ( A ) Immunocytochemical analysis of primary cultures of the dorsal thalamus using Ca V 1.2- (red), AKAP150- (green) and PKARIIβ- (blue) specific antibodies. Merged picture shows the close connection of the components of the proposed ternary complex, especially in somatic regions and proximal dendrites. Enlarged inlay represents a magnification of the area indicated by the rectangle. Data shown are representative pictures from several independent immunostainings and preparations of neurons. In all cases, omission of primary antibodies resulted without signal (negative control). Western blot analysis and pull down assays were done as described in " ". ( B ) Interaction of AKAP7-MBP and PKARIIβ-c-myc was detected using antibodies against c-myc. ( C ) IP of PKARIIβ-c-myc and AKAP5-GFP detected after incubation with GFP-coupled magnetic beads using antibodies derived against PKARIIβ. ( D ) Existence of PKA holoenzyme consisting of PKARIIβ-GST and PKAcsβ-GFP was detected with antibodies against GFP protein.

Article Snippet: After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-Ca V 1.2 (1∶200, Alomone Labs, Israel); mouse anti-cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIβ, 1∶500, BD Bioscience, USA); rabbit anti-β 2 -AR (H-73 1∶400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1∶1000, Sigma, Germany); goat anti-AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phosphatase 2A (PP2A, Acris, Germany).

Techniques: Negative Control, Western Blot, Incubation, Magnetic Beads, Derivative Assay

Journal: PLoS ONE

Article Title: Modulation of Calcium-Dependent Inactivation of L-Type Ca 2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

doi: 10.1371/journal.pone.0027474

Figure Lengend Snippet: ( A ) Representative current traces recorded under control conditions (upper left panel) and in the presence of okadaic acid (OA) (10 µM; left down panel). The bar graph (right panel) represents D inact under different recording conditions (as indicated). The mean value of three cells recorded under control conditions was taken for comparison with three cells recorded under 10 µM OA. ( B ) Representative current traces recorded under control conditions (upper left panel) and in the presence of the isoproterenol alone (10 µM; left middle panel) or in combination with OA (10 µM; lower left panel). The bar graph (right panel) represents D inact under different recording conditions (as indicated). The mean value of five cells recorded under control conditions was taken for comparison with five cells recorded under 10 µM isoproterenol and five cells recorded under isoproterenol plus OA. Data are presented as means ± SEM of several independent experiments. *** P <0.001. Significance of isoproterenol plus OA (n = 5) versus controls (n = 5) was calculated by Student's t test. ** P <0.01. Significance of isoproterenol (n = 5) versus isoproterenol plus OA (n = 5) was calculated by Student's t test. The degree of inactivation is given by the normalized current amplitude of the mean postpulse I/V at +10 mV. ( C ) Immunocytochemical analysis of primary cultures of the dorsal thalamus using Ca V 1.2- (left panel, green) and PP2A-specific antibodies (right panel, red). Merge picture showed close association of the two proteins, especially in somatic regions and proximal dendrites. Data shown are representative pictures from several independent immunostainings and thalamic neurons preparations. In all cases, omission of primary antibodies resulted in no fluorescence signal above background (negative control).

Article Snippet: After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-Ca V 1.2 (1∶200, Alomone Labs, Israel); mouse anti-cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIβ, 1∶500, BD Bioscience, USA); rabbit anti-β 2 -AR (H-73 1∶400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1∶1000, Sigma, Germany); goat anti-AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phosphatase 2A (PP2A, Acris, Germany).

Techniques: Fluorescence, Negative Control

Journal: Nature Communications

Article Title: Selective posttranslational inhibition of Ca V β 1 -associated voltage-dependent calcium channels with a functionalized nanobody

doi: 10.1038/s41467-022-35025-7

Figure Lengend Snippet: a Cartoon depicting hypothesized association of Ca V 1.2 with β 2b subunits in adult ventricular myocytes (i) and the expected loss of β 2b with gene knockout of this subunit (ii). Bottom, knock out of β 2 in adult heart has only minimal impact on whole-cell Ca 2+ current making it ambiguous whether most Ca V 1.2 are associated with β 2b in adult ventricular cardiomyocytes. Posttranslational inhibition of cardiac Ca V 1.2 using a Ca V β-targeted nanobody fused to Nedd4L HECT (Ca V -aβlator) domain eliminates Ca V 1.2 complexes from the membrane and abolishes current (iii), proving that Ca V 1.2 is stably associated with β 2 in adult cardiomyocytes. b Schematic of a scenario where distinct Ca V β isoforms preferentially associate with particular α 1 -subunit types to mediate different functions (i). Knockdown of one β-isoform may lead to β reshuffling that lessens the functional impact of elimination of the particular Ca V β subunit (ii). By contrast, targeted posttranslational inhibition of the channel complex based on the β isoform would yield a qualitatively different result that more accurately reflects the functional logic of Ca V β molecular diversity in the cell (iii).

Article Snippet: Cells were then incubated with rabbit anti-Ca V 1.2 primary antibody (Alomone Labs, 1:1000) in PBS containing 1% NGS, 1% BSA, and 0.1% BSA overnight at 4 °C.

Techniques: Gene Knockout, Knock-Out, Inhibition, Stable Transfection, Functional Assay

Journal: Nature Communications

Article Title: Selective posttranslational inhibition of Ca V β 1 -associated voltage-dependent calcium channels with a functionalized nanobody

doi: 10.1038/s41467-022-35025-7

Figure Lengend Snippet: a Schematic of skeletal muscle Cav1.1 complex. b Images of isolated flexor digitorum brevis (FDB) fibers either untransfected (top) or transfected with Chisel-1-P2A-CFP (bottom). c Top, exemplar whole-cell currents from isolated FDB fibers expressing CFP (left) or Chisel-1 (right). Bottom, Population J-V curves from isolated FDB fibers expressing CFP (black squares; n = 13 over 3 independent experiments) or Chisel-1 (red squares; n = 13 over 3 independent experiments). d Top, exemplar gating currents from isolated FDB fibers expressing CFP (left) or Chisel-1 (right). Bottom, Population Q-V curves from isolated FDB fibers expressing CFP (black circles; n = 7 over 2 independent experiments) or Chisel-1 (red circles; n = 8 over 2 independent experiments). * P = 9.32 × 10 −5 compared to CFP control, two-tailed unpaired t test. e Schematic of ventricular cardiomyocyte Ca V 1.2 complex. f Confocal images of cardiomyocytes expressing mCherry ( top ) or Chisel-1-P2A-mCherry (bottom). g Population J-V curves from isolated ventricular myocytes expressing mCherry (black triangles; n = 13 over 3 independent experiments) or Chisel-1 (red triangles; n = 11 over 3 independent experiments). h Top, exemplar whole-cell currents from ventricular myocytes expressing mCherry (left) or Chisel-1 (right) before (black) and after (cyan) application of 1 μM forskolin. Bottom, lack of effect of Chisel-1 on forskolin induced increase in I Ca,L in ventricular myocytes (mCherry, n = 14; Chisel-1, n = 5). Data are means ± SEM. Source data are provided as a Source Data file.

Article Snippet: Cells were then incubated with rabbit anti-Ca V 1.2 primary antibody (Alomone Labs, 1:1000) in PBS containing 1% NGS, 1% BSA, and 0.1% BSA overnight at 4 °C.

Techniques: Isolation, Transfection, Expressing, Two Tailed Test

Journal: PLoS ONE

Article Title: The Impact of Ovariectomy on Calcium Homeostasis and Myofilament Calcium Sensitivity in the Aging Mouse Heart

doi: 10.1371/journal.pone.0074719

Figure Lengend Snippet: A . Detection of ~240 kDa bands corresponding to Ca V 1.2 in ventricular tissue from sham and OVX mice. Na/K ATPase (110 kDa band) was used as a loading control (15 µg of protein were loaded in each lane). Average Ca V 1.2 band intensity was lower in OVX ventricular tissue compared to sham controls (n=3 hearts/group). B . Representative immunoblot illustrating ~116 kDa bands corresponding to NCX in sham and OVX ventricle. The loading control was Na-K ATPase as in A. Mean normalized NCX band intensity was similar in ventricular tissue from sham and OVX mice (n=3 hearts in each group). C . Detection of ~110 kDa bands corresponding to SERCA2 in the ventricles of sham and OVX mice. Amido black was used as a loading control (60 µg of protein were loaded in each lane). Average intensity of the SERCA2 bands was similar in sham and OVX ventricles (n=3 hearts in each group). In experiments where Na-K ATPase was used as a loading control, there was no significant difference in Na-K ATPase protein levels between sham and OVX (t-test, p=0.164) (*denotes p<0.05; t-test)..

Article Snippet: Antibodies used were rabbit anti- Ca v 1.2 polyclonal antibody (Alomone, ACC-003-AG; 1:2000), mouse anti-NCX monoclonal antibody (SWANT, R3F1; 1:1000), mouse anti-SERCA2 monoclonal antibody (Affinity Bioreagents, MA3-919; 1:2000) and rabbit anti-Na/KATPase polyclonal antibody (Abcam; 1:2000).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: The Impact of Ovariectomy on Calcium Homeostasis and Myofilament Calcium Sensitivity in the Aging Mouse Heart

doi: 10.1371/journal.pone.0074719

Figure Lengend Snippet: Comparison of Key Ca 2+ Handling Mechanisms in Hearts and Cardiomyocytes From Young Adult OVX and Aged OVX Female Mice.

Article Snippet: Antibodies used were rabbit anti- Ca v 1.2 polyclonal antibody (Alomone, ACC-003-AG; 1:2000), mouse anti-NCX monoclonal antibody (SWANT, R3F1; 1:1000), mouse anti-SERCA2 monoclonal antibody (Affinity Bioreagents, MA3-919; 1:2000) and rabbit anti-Na/KATPase polyclonal antibody (Abcam; 1:2000).

Techniques: In Vivo, Activity Assay, Expressing

Journal: Cardiovascular Therapeutics

Article Title: LCZ696 Therapy Reduces Ventricular Tachyarrhythmia Inducibility in a Myocardial Infarction-Induced Heart Failure Rat Model

doi: 10.1155/2019/6032631

Figure Lengend Snippet: Protein expression.

Article Snippet: The membranes were then incubated in Tween-TBS with the primary antibodies, including a rabbit anti-Na V 1.5 (AVIVA Systems Biology, San Diego, CA, US), a rabbit anti- Ca V 3.1 antibody (Abcam, Cambridge, UK), a rabbit anti- Ca V 1.2 antibody (Alomone labs, Jerusalem, Israel), a rabbit anti-K V 7.1 antibody (Alomone labs, Jerusalem, Israel), a mouse anti-K V 4.3 antibody (Abcam, Cambridge, UK), a rabbit anti-ERG antibody (Abcam, Cambridge, UK), a rabbit anti-KCNE1 antibody (Proteintech, Chicago, IL, US), a rabbit anti-KCNE2 antibody (Alomone labs, Jerusalem, Israel), a rabbit anti-CX43 antibody (Proteintech, Chicago, IL, US), and a rabbit anti-tubulin antibody (Sigma, St. Louis, MO, US).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Novel Pannexin-1-Coupled Signaling Cascade Involved in the Control of Endothelial Cell Function and NO-Dependent Relaxation

doi: 10.1155/2021/2678134

Figure Lengend Snippet: Expression of voltage-dependent Na + (Na v ) and Ca 2+ (Ca v ) channels in endothelial cells of mesenteric resistance arteries. (a) Immunohistochemistry analysis of the cellular distribution of Na v and Ca v channel-specific isoforms Na v 1.2, Na v 1.6, and Ca v 3.2 in the wall of mesenteric resistance arteries. Note that Na v 1.2 and Na v 1.6 channels are present in both endothelial cells and smooth muscle cells, but Ca v 3.2 channels are expressed exclusively in the endothelium. Arrows highlight the staining observed in endothelial cells. (b) Immunofluorescence detection of the expression of Na v 1.2, Na v 1.6, and Ca v 3.2 channels in primary cultures of mesenteric endothelial cells. (c) Representative Western blots and densitometric analysis of the expression of eNOS and Na v 1.2, Na v 1.6, and Ca v 3.2 channels in intact mesenteric arteries before (E + ) and after (E − ) removing the endothelium by the treatment with collagenase.

Article Snippet: After blocking the endogenous peroxidase activity, sections were incubated overnight at 4°C with anti-Na v 1.2, anti-Na v 1.6, or anti-Ca v 3.2 rabbit primary antibodies (Alomone Laboratories, Israel) and the signal was developed using the biotin link secondary antibody (10 min), HPR label, and DAB chromogen of the Mouse/Rabbit ImmunoDetector System.

Techniques: Expressing, Immunohistochemistry, Staining, Immunofluorescence, Western Blot