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: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

doi: 10.1073/pnas.2207887120

Figure Lengend Snippet: Ca(v) 1.2 α1C subunit is palmitoylated in mouse, rabbit, and human ventricular tissues. Palmitoylated proteins were purified by resin-assisted capture of acylated proteins (acyl-RAC) and immunoblotted as shown. The bar chart below each blot indicates the abundance of Ca(v)1.2 α1C and caveolin 3 (Cav3) in the purified palmitoylated fraction (Palm) relative to the corresponding unfractionated lysate (UF). N = 4 (mouse), N = 8 (rabbit), N = 7 (human). *** P < 0.001, unpaired t test. Error bars represent SEM.

Article Snippet: Anti-Ca(v)1.2 α1C subunit antibodies raised in rabbit and guinea pig were obtained from Alomone Labs, antibodies against flotillin 2 and caveolin 3 from BD Biosciences, and anti-GFP antibodies from Abcam and Protein Tech.

Techniques: Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes

doi: 10.1073/pnas.2207887120

Figure Lengend Snippet: Palmitoylation site conservation in human Ca(v)1 channel isoforms. For clarity, regions of the rabbit α1C splice variant of Ca(v)1.2 CACH2A (UniProt accession number P15381) with palmitoylated cysteines identified in this investigation numbered and highlighted in red are shown above the corresponding regions of the human channels. Numbers at the end of each sequence are the numbering of the final amino acid in the region of each Ca(v)1 isoform shown. Human Ca(v)1 isoforms (UniProt accession numbers shown) were aligned using Clustal Ω. “*” below an amino acid indicates 100% conservation between isoforms; “:” indicates amino acids of highly similar properties; “.” indicates amino acids of weakly similar properties. The palmitoylation site in the Ca(v)1.2 N terminus is conserved in all isoforms. Ca(v)1.1 does not possess a cysteine analogous to C519 in the I–II linker, but Ca(v)1.3 and 1.4 do. C543 is unique to Ca(v)1.2.

Article Snippet: Anti-Ca(v)1.2 α1C subunit antibodies raised in rabbit and guinea pig were obtained from Alomone Labs, antibodies against flotillin 2 and caveolin 3 from BD Biosciences, and anti-GFP antibodies from Abcam and Protein Tech.

Techniques: Variant Assay, Sequencing

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: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Sequence of primers and PCR conditions for the different subunits of L-VDCC.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Sequencing, Amplification

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Detection of mRNA for L-VDCC subunits in guinea pig tracheal smooth muscle, as revealed by RT-PCR. (a) In airway smooth muscle, the PCR products at 470 and 459 bp length correspond to Ca V 1.2 and Ca V 1.3 cDNA, respectively. In this tissue, Ca V 1.1 and Ca V 1.4 were not found. Positive controls for these subunits were skeletal muscle (SKM, ~500 bp) and retina (~200 bp). Lane at the left corresponds to 1 Kb Plus DNA Ladder. (b) Representative PCR blots for Ca V 1.2 and Ca V 1.3 from nonsensitized (NS, n = 3) and sensitized (S, n = 4) smooth muscles. The first column in each blot corresponds to a negative control without template. The lower panel displays constitutive cDNA of GAPDH. (c) Densitometry data analysis for mRNA from Ca V 1.2 and Ca V 1.3 subunits showing no statistical significance between NS and S. Bars correspond to mean ± SEM.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Immunofluorescence for Ca V 1.2 in nonsensitized and sensitized guinea pig tracheal smooth muscle. The first column shows immunoreactivity for Ca V 1.2 (stained green) in nonsensitized (a) and sensitized tissues (e); notice that Ca V 1.2 is located in the airway smooth muscle (SM) and epithelium (EPI, pointed by arrow); blocking peptide completely eliminated the fluorescence (i). The second and the third columns illustrate smooth muscle α -actin (stained red; (b), (f), (j)) and cell nuclei (DAPI, stained blue; (c), (g), (k)). The last column depicts merged images of the former three columns ((d), (h), (l)). In these merged images, Ca V 1.2 is seen to be colocalized with α -actin (stained yellow) on the smooth muscle.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Immunofluorescence, Staining, Blocking Assay, Fluorescence

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Sequence of primers and PCR conditions for the different subunits of L-VDCC.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Sequencing, Amplification

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Detection of mRNA for L-VDCC subunits in guinea pig tracheal smooth muscle, as revealed by RT-PCR. (a) In airway smooth muscle, the PCR products at 470 and 459 bp length correspond to Ca V 1.2 and Ca V 1.3 cDNA, respectively. In this tissue, Ca V 1.1 and Ca V 1.4 were not found. Positive controls for these subunits were skeletal muscle (SKM, ~500 bp) and retina (~200 bp). Lane at the left corresponds to 1 Kb Plus DNA Ladder. (b) Representative PCR blots for Ca V 1.2 and Ca V 1.3 from nonsensitized (NS, n = 3) and sensitized (S, n = 4) smooth muscles. The first column in each blot corresponds to a negative control without template. The lower panel displays constitutive cDNA of GAPDH. (c) Densitometry data analysis for mRNA from Ca V 1.2 and Ca V 1.3 subunits showing no statistical significance between NS and S. Bars correspond to mean ± SEM.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Mediators of Inflammation

Article Title: Tumor Necrosis Factor Alpha Inhibits L-Type Ca 2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

doi: 10.1155/2016/5972302

Figure Lengend Snippet: Immunofluorescence for Ca V 1.3 in nonsensitized and sensitized guinea pig tracheal smooth muscle. The first column shows immunoreactivity for Ca V 1.3 (stained green) in nonsensitized (a) and sensitized tissues (e); notice that Ca V 1.3 is located in the airway smooth muscle (SM) and epithelium (EPI, pointed by arrows); blocking peptide completely eliminated the fluorescence (i). The second and the third columns illustrate smooth muscle α -actin (stained red; (b), (f), (j)) and cell nuclei (DAPI, stained blue; (c), (g), (k)). The last column depicts merged images of the former three columns ((d), (h), (l)). In these merged images, Ca V 1.3 is seen to be colocalized with α -actin (stained yellow) on the smooth muscle.

Article Snippet: To block nonspecific binding to proteins, 10% horse serum was applied on the slices for two h. The slices were incubated with the primary antibodies to Ca V 1.2 and Ca V 1.3 (subunits of L-VDCC, Alomone Labs., Cat. numbers ACC-003 and ACC-311, resp., Jerusalem, Israel), both antibodies at a dilution 1 : 50, overnight at 4°C.

Techniques: Immunofluorescence, Staining, Blocking Assay, Fluorescence

Journal: PLoS ONE

Article Title: PI3Ks Maintain the Structural Integrity of T-Tubules in Cardiac Myocytes

doi: 10.1371/journal.pone.0024404

Figure Lengend Snippet: ( a ) Confocal microscopy images of myocytes from control and dKO mice colabeled with antibodies against Ca V 1.2 (green) and RyR (red). ( b ) Pearson's coefficient for colocalization of Ca V 1.2 and RyR ( n = 19 cells from ≥3 hearts per group). Data shown are mean ± SEM (* P = 8.1×10 −9 , t -test). 1D Fourier analysis of spatial frequency for Ca V 1.2 ( c ) and RyR ( d ) along the longitudinal axis of representative myocytes (averaged value from >10 lines). ( e ) The normalized output of 1D Fourier transform analysis from 19–20 cells for each group. Data shown are mean ± SEM (* P = 0.0001 (Ca V 1.2) and 0.015 (RyR), t -test). ( f ) Representative ind-dKO and ind-control myocytes colabeled with antibodies against Ca V 1.2 (green) and RyR (red). ( g ) Pearson's coefficient for colocalization of Ca V 1.2 and RyR ( n = 13–16 cells from ≥3 hearts per group). Data shown are mean ± SEM (* P = 0.016, t -test). 1D Fourier analysis of spatial frequency for Ca V 1.2 ( h ) and RyR ( i ) along the longitudinal axis of representative myocytes (averaged value from >10 lines). ( j ) The normalized output of 1D Fourier transform analysis from 19 cells for each group. Data shown are mean ± SEM (* P = 9.8×10 −6 (Ca V 1.2) and 4.3×10 −7 (RyR), t -test).

Article Snippet: Ca V 1.2 antibody was from Alomone Labs. RyR antibody was from Affinity Bioreagents.

Techniques: Confocal Microscopy

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: Molecular Pain

Article Title: Familial hemiplegic migraine Ca V 2.1 channel mutation R192Q enhances ATP-gated P2X 3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

doi: 10.1186/1744-8069-6-48

Figure Lengend Snippet: Ca 2+ transients evoked by K + depolarization or P2X 3 receptors in WT and R192Q KI neurons . A , Examples of Ca 2+ transients of trigeminal neurons evoked by KCl (20 mM, 2-s application) before ( black trace ) and after ( red trace ) application of ω-agatoxin (200 nM, 30 min). B , KI neurons show significant increase in KCl (20 mM, 2-s application) mediated Ca 2+ transients compared to WT (* p = 0.005, n = 28 and n = 45, in WT and KI, respectively). Histograms also represent inhibition by ω-agatoxin of Ca 2+ transients for WT ( n = 14) and KI ( n = 35) neurons. After ω-agatoxin responses of WT and KI neurons differ from their own controls (** p ≤ 0.001). C , Representative traces of α,β-meATP (10 μ M, 2-s application)-evoked Ca 2+ transients before ( black trace ) and after ( red trace ) application of ω-agatoxin (200 nM, 30 min). D , Histograms show larger Ca 2+ transients evoked by α,β-meATP (10 μ M, 2-s application) from KI ( n = 26) than WT ( n = 16) and neurons (* p = 0.04). Histograms also show that ω-agatoxin reduced Ca 2+ transients of KI ( n = 22) and WT ( n = 9) neurons. ** p ≤ 0.001 for each case. E , Microphotographs of immunofluorescence experiments depicting WT and KI trigeminal neurons in culture expressing P2X 3 receptors or Ca V 2.1 channels. Bar = 50 μ m. Histograms ( right ) show% of P2X 3 - (top) or Ca V 2.1- (bottom) immunoreactive neurons (taking as 100% the β-tubulin III immunoreactive) ( n = 5, p > 0.05 for P2X 3 receptors; n = 3, p > 0.05 for Ca V 2.1-expressing neurons). F , Histograms show% of Ca V 2.1-immunoreactive neurons ( top ; taken as 100%) which are immunopositive for P2X 3 ( n = 7, p > 0.05) or% of P2X 3 -immunoreactive neurons ( bottom ) which are immunopositive for Ca V 2.1 (n = 4, p > 0.05).

Article Snippet: The following antibodies were used: anti-P2X 3 (1:300; Alomone Labs), anti-P2X 3 (H-60, 1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-cyclin dependent kinase 5 (Cdk5; 1: 400; Santa Cruz), anti-p35 (1:200, Santa Cruz), anti-Ca V 2.1 α1 (1:200; Alomone Labs), anti-phosphorylated tyrosine HRP-conjugated clone Y20 (1:3,000; Invitrogen), anti-phospho-serine (1:600; Millipore), anti-phosphorylated threonine (1:600; Cell Signaling Danvers, MA, USA), anti-β-tubulin III (1:2,000; Sigma), anti-actin (1:3,000: Sigma).

Techniques: Inhibition, Immunofluorescence, Expressing

Journal: Molecular Pain

Article Title: Familial hemiplegic migraine Ca V 2.1 channel mutation R192Q enhances ATP-gated P2X 3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

doi: 10.1186/1744-8069-6-48

Figure Lengend Snippet: Survival of WT and KI trigeminal neurons in culture and their expression of Ca v 2.1 protein . A , Survival is calculated as number of β-tubulin III positive cells per unit area after 1-4 days in culture. Data are normalized with respect to those at 1 day. n = 4, p > 0.05. B , Somatic size distribution of trigeminal neurons (β-tubulin III immunoreactive) in culture from WT and KI mice. n = 4. C , Immunocytochemical expression of Ca V 2.1 channels in intact trigeminal ganglia of WT and KI mice. Histograms represent% of Ca V 2.1 immunoreactive neurons over β-tubulinIII immunoreactive neurons in WT or KI ganglia. n = 3, p > 0.05. D , Example of western blot of protein extracts from WT and KI trigeminal ganglia or culture, probed with anti-Ca V 2.1 antibody. Equal loading was ensured by membrane probing with β-tubulinIII antibodies. n = 3, p > 0.05. Histograms ( right ) show no significant difference between these conditions.

Article Snippet: The following antibodies were used: anti-P2X 3 (1:300; Alomone Labs), anti-P2X 3 (H-60, 1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-cyclin dependent kinase 5 (Cdk5; 1: 400; Santa Cruz), anti-p35 (1:200, Santa Cruz), anti-Ca V 2.1 α1 (1:200; Alomone Labs), anti-phosphorylated tyrosine HRP-conjugated clone Y20 (1:3,000; Invitrogen), anti-phospho-serine (1:600; Millipore), anti-phosphorylated threonine (1:600; Cell Signaling Danvers, MA, USA), anti-β-tubulin III (1:2,000; Sigma), anti-actin (1:3,000: Sigma).

Techniques: Expressing, Western Blot