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: Acta Histochemica et Cytochemica

Article Title: Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca 2+ Channels in Rat Adrenal Medullary Chromaffin Cells

doi: 10.1267/ahc.23-00042

Figure Lengend Snippet: Different distribution of Ca V 1.2 channel and muscarinic M 4 receptor in the cell membrane in rat AMC cells. ( A ) Immunostaining of Ca V 1.2-YFP fusion protein expressed in HEK293T cells with a rabbit anti-Ca V 1.2 antibody (Ab). HEK293T transfected with a Ca V 1.2-YFP construct were labeled with the rabbit anti-Ca V 1.2 Ab. a and b represent confocal images of Ca V 1.2-like immunofluorescence and YFP fluorescence, respectively; c represents a differential interference contrast (DIC) image. The immunoreaction and YFP fluorescence were visualized with excitation at 514 nm and emission of 530–600 nm and with excitation at 633 and emission above 650 nm, respectively. ( B ) Fractionation analysis of rat adrenal medullae for integral membrane proteins. The cell membrane was divided into the raft and non-raft membrane domains by using discontinuous sucrose density gradient centrifugation (see the Materials and Methods). The same volume of each fraction with 5%–40% sucrose was immunoblotted for caveolin-1, transferrin receptor (R), muscarinic M 4 receptor, and TASK1 channel. Note that caveolin-1, a raft membrane marker, was enriched in the 20% fraction, whereas transferrin R, a non-raft membrane marker, was present in the 40% fraction. ( C ) Double staining for caveolin-1 and Ca V 1.2 and for M 4 receptor and Ca V 1.2 in rat AMC cells. The first column indicates confocal images of caveolin-1 and M 4 receptor-like immunofluorescence. The second column shows confocal images of Ca V 1.2-like immunofluorescence. The third column is a merge of immunofluorescence images. The fourth column shows DIC images. The calibration applies to all the images. Dissociated rat AMC cells were treated overnight with rabbit anti-Ca V 1.2 Ab (dilution, 1:50) and mouse anti-caveolin-1 Ab (1:20) or mouse anti-M 4 Ab (1:50). Ca V 1.2 and caveolin-1 or M 4 receptor-like immunoreactive material were visible as rhodamine and FITC-like fluorescence, respectively. ( D ) Summary of the coincidence rates of caveolin-1 (Cav1) and M 4 with Ca V 1.2. The data represent the mean ± SEM (Cav1/Ca V 1.2, n = 10; M 4 /Ca V 1.2, n = 5). Statistical significance was evaluated with an unpaired Student’s t test.

Article Snippet: First, it was incubated with one of the following primary antibodies (Abs): rabbit anti-caveolin-1 (sc-894: Santa Cruz Biotechnology, Santa Cruz, CA, USA) (RRID:AB_2072042), mouse anti-transferrin receptor (A11130: Molecular Probes, Eugene, OR, USA) (RRID:AB_2534136), mouse anti-M 4 (MAB1576: Chemicon, Temecula, CA, USA) (RRID:AB_2080217), rabbit anti-Ca V 1.2 (ACC-001: Alomone, Jerusalem, Israel) (RRID:AB_2039764), or rabbit anti-TWIK-related acid-sensitive K + 1 (TASK1) (APC-024: Alomone) (RRID:AB_2040132).

Techniques: Membrane, Immunostaining, Transfection, Construct, Labeling, Immunofluorescence, Fluorescence, Fractionation, Gradient Centrifugation, Marker, Double Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Muscarinic Receptor Stimulation Does Not Inhibit Voltage-dependent Ca 2+ Channels in Rat Adrenal Medullary Chromaffin Cells

doi: 10.1267/ahc.23-00042

Figure Lengend Snippet: Diagram showing localization of caveolin-1, Ca V 1.2, muscarinic M 4 receptor subtype, and TASK1 in the raft and non-raft membrane domains.

Article Snippet: First, it was incubated with one of the following primary antibodies (Abs): rabbit anti-caveolin-1 (sc-894: Santa Cruz Biotechnology, Santa Cruz, CA, USA) (RRID:AB_2072042), mouse anti-transferrin receptor (A11130: Molecular Probes, Eugene, OR, USA) (RRID:AB_2534136), mouse anti-M 4 (MAB1576: Chemicon, Temecula, CA, USA) (RRID:AB_2080217), rabbit anti-Ca V 1.2 (ACC-001: Alomone, Jerusalem, Israel) (RRID:AB_2039764), or rabbit anti-TWIK-related acid-sensitive K + 1 (TASK1) (APC-024: Alomone) (RRID:AB_2040132).

Techniques: Membrane

Journal: Scientific Reports

Article Title: A novel binding site between the voltage-dependent calcium channel Ca V 1.2 subunit and Ca V β2 subunit discovered using a new analysis method for protein–protein interactions

doi: 10.1038/s41598-023-41168-4

Figure Lengend Snippet: Figure 2 (a) Strategy for creating a Ca V 1.2 epitope library with the β2 subunit. Full-length cDNA of Ca V β2 gene is in the arabinose unit and the Ca V 1.2 epitope library is in the IPTG unit. ( b ) Construction of a Ca V 1.2 epitope-library (IPTG-sensitive toxin-in-frame clone library). ( i ) Blunt-ended DNA fragment from Ca V 1.2 is inserted at the Sma I site (with a single frameshift with the first iUnit) of the IPTG unit. ( ii ) Clones with the first iUnit (first library). ( iii ) First iUnit in the library is deleted using Spe I and re-ligated. Only clones with the chimeric gene and neomycin-resistance gene form colonies (second library). ( iv ) Neomycin-resistance gene is eliminated using Xho I and re-ligated (third library).

Article Snippet: The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca V 1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca V 1.2.

Techniques: Clone Assay

Journal: Scientific Reports

Article Title: A novel binding site between the voltage-dependent calcium channel Ca V 1.2 subunit and Ca V β2 subunit discovered using a new analysis method for protein–protein interactions

doi: 10.1038/s41598-023-41168-4

Figure Lengend Snippet: Results of third screening of pdGENE-Toxin sensitivity assay. ( a ) Candidate clones with selection. Selection by ampicillin, arabinose, and IPTG, or ampicillin and arabinose. #23, #24, #30, #31, #32, #36, #37 and #46 form colonies under ampicillin/arabinose/IPTG and ampicillin/arabinose. Negative control was clone #2, which was IPTG-sensitive (negative selection). Sequence direction and corresponding sequences are indicated. Clones in the antisense direction (#23, 32, and 36) and those with an E. coli -derived sequence were eliminated. Clone #37 was eliminated because it contained a Ca V 1.2 transmembrane region. ( b ) Overlapping sequence of clones 24 and 31. Translated amino acid sequences are above nucleotide sequences (single-letter code) and numbered.

Article Snippet: The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca V 1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca V 1.2.

Techniques: Sensitive Assay, Clone Assay, Selection, Negative Control, Sequencing, Derivative Assay

Journal: Scientific Reports

Article Title: A novel binding site between the voltage-dependent calcium channel Ca V 1.2 subunit and Ca V β2 subunit discovered using a new analysis method for protein–protein interactions

doi: 10.1038/s41598-023-41168-4

Figure Lengend Snippet: Coimmunoprecipitation analysis of interactions between the β2 subunit and Ca V 1.2-derived clone (24) in human embryonic kidney 293 (HEK-293) cells. ( a ) Western blot analyses of Ca V β2 in HEK-293 T cells. Transected constructs are indicated. Lane, 1 Ca V β2; lane 2, Ca V β2 and EGFP-24; lane 3, Ca V β2 and full-length Ca V 1.2. ( b ) Coimmunoprecipitation analysis of EGFP-24 and Ca V β2. An anti-GFP antibody revealed a single band that corresponded to the EGFP-24 fusion protein (lane 2). ( c ) Coimmunoprecipitation analysis of Ca V 1.2 and Ca V β2. An anti-Ca V 1.2 antibody revealed a single band (lane 3, 200 kDa).

Article Snippet: The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca V 1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca V 1.2.

Techniques: Derivative Assay, Western Blot, Construct

Journal: Scientific Reports

Article Title: A novel binding site between the voltage-dependent calcium channel Ca V 1.2 subunit and Ca V β2 subunit discovered using a new analysis method for protein–protein interactions

doi: 10.1038/s41598-023-41168-4

Figure Lengend Snippet: ( a ) Amino acid sequence of the C-terminus of Ca V 1.2. The IQ domain (light green), CAC1F_C domains (red), C-terminal binding site (CBS), distal C-terminal regulatory domain (black underlined), leucine-zipper-like region (red asterisks), and CaM-competitive domain (green underlined) were obtained from the NCBI database ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). ( b ) Schematic of Ca V 1.2. The four transmembrane domains are labelled I to IV. The high-affinity interaction domain (α-interaction domain; AID) is located between domains I and II. The IQ domain, CAC1F domain, and C-terminal binding site (CBS) are located at the C-terminus.

Article Snippet: The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca V 1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca V 1.2.

Techniques: Sequencing, Binding Assay

Journal: Scientific Reports

Article Title: A novel binding site between the voltage-dependent calcium channel Ca V 1.2 subunit and Ca V β2 subunit discovered using a new analysis method for protein–protein interactions

doi: 10.1038/s41598-023-41168-4

Figure Lengend Snippet: ( a ) Protein alignment of the C-terminal binding sites (CBS) of rabbit α1 subunits. Amino acid sequence alignments of Ca V 1.1, Ca V 1.2, Ca V 1.3, Ca V 1.4, Ca V 2.1, Ca V 2.2, Ca V 2.3, and Ca V 3.1 obtained using ClustalW ( https://www.genome.jp/tools-bin/clustalw ). The CBS in Ca V 1.2 is in red. Protein IDs are indicated. *, identity; -, gap. Alignment of the CBS of rabbit Ca V 1.2 and Ca V 1.3 (inset). CBS sequence of Ca V 1.3 shows homology with that of Ca V 1.2. ( b ) Amino acid alignment of the CBS of Ca V 1.2 in different species. CBS sequences of zebrafish, xenopus, human, rabbit, mouse, and rat were aligned using ClustalW ( https://www.genome.jp/tools-bin/clustalw ). CBS in rabbit Ca V 1.2 is in red. Protein IDs are indicated. *, identity; -, gap.

Article Snippet: The eluted product (15 μL) and an equal volume of whole-cell lysate were subjected to 10% SDS-PAGE and Western blotting (ProBlot II AP; Promega) with an anti-β2 (Sigma-Aldrich), anti-GFP (GeneTex Inc., Irvine, CA), or anti-Ca V 1.2 (Alomone, Jerusalem, Israel) polyclonal antibody, which recognizes the intracellular loop between domains II and III of Ca V 1.2.

Techniques: Binding Assay, Sequencing

Journal: Nature Communications

Article Title: NPC1-dependent alterations in K V 2.1–Ca V 1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease

doi: 10.1038/s41467-023-39937-w

Figure Lengend Snippet: A Left: Representative super-resolution Airyscan images taken at a focal plane near the plasma membrane (PM) from CTL (black) and U18-treated (red) neurons fixed and immunolabeled for Ca V 1.2. Right , quantification of PM Ca V 1.2 total intensity, cluster density and cluster size of CTL (black) and U18-treated (red) neurons in the soma (left, yellow) and dendrite (right, orange) regions. N = 46–48 (CTL) and n = 58–59 (U18) neurons, and n = 145 (CTL) and n = 161 (U18) dendrites were analyzed across 5 independent isolations. B Left , representative super-resolution TIRF localization maps of CTL (black) and U18-treated (red) neurons fixed and immunolabeled for Ca V 1.2. Right , quantification of PM Ca V 1.2 cluster density, cluster size, and nearest cluster distance of CTL (black) and U18-treated (red) neurons in the soma region. N = 20 (CTL) and n = 21 (U18) neurons were analyzed across 3 independent isolations. C Same as ( A ) only neurons fixed and immunolabeled for Ca V 1.3. N = 32 (CTL) and n = 38 (U18) neurons, and n = 104 (CTL) and n = 123 (U18) dendrites were analyzed across 3 independent isolations. D Same as B, only immunolabeled for Ca V 1.3. N = 16 (CTL) and n = 18 (U18) neurons were analyzed across 2 independent isolations. All error bars represent SEM. Statistical significance was calculated using Mann–Whitney (two-tailed) and Unpaired t tests (two-tailed) in ( A )–( D ). ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. CTL is control and U18 is U18666A.

Article Snippet: After 30 min incubation at RT in Tris-Buffered Saline (TBS) buffer supplemented with 0.05% Tween‐20 (TBS-T) and 5% non‐fat dry milk, membranes were exposed O/N at 4 °C to the following primary antibodies: K V 2.1 at 4 μg/mL (NeuroMab, K89/34), Ca V 1.2 at 1:250 (Alomone Labs, acc-003), CDK5 at 1:100 (Santa Cruz Biotechnology, Cat #sc-6247), p35 at 1:100 (Cell Signaling Technology, Cat #C64B10), p39 at 1:100 (Santa Cruz Biotechnology, Cat #sc-365781) or GAPDH at 1:1000 (Proteintech, Cat #10494-I-AP).

Techniques: Immunolabeling, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: NPC1-dependent alterations in K V 2.1–Ca V 1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease

doi: 10.1038/s41467-023-39937-w

Figure Lengend Snippet: A Top , representative super-resolution TIRF images of CTL (black) and U18-treated (red) neurons co-immunolabeled for Ca V 1.2 and K V 2.1. Bottom , quantification of % of soma occupied by K V 2.1–Ca V 1.2, K V 2.1–Ca V 1.2 cluster density and K V 2.1–Ca V 1.2 nearest cluster distance of CTL (black) and U18-treated (red) neurons in the soma region. N = 9 (CTL) and n = 10 (U18) neurons were analyzed across 2 independent isolations. B Top , representative Ca V 1.2 and K V 2.1 PLA images of CTL (black) and U18-treated (red) neurons. Bottom , quantification of PLA puncta density and nearest puncta distance of CTL (black) and U18-treated (red) neurons. N = 19 (CTL) and n = 17 (U18) neurons were analyzed across 2 independent isolations. C Left , representative maximum intensity projections from WT (black) and NPC1 I1061T (red) cerebellar sagittal sections co-immunolabeled for Calbindin, Ca V 1.2 and K V 2.1. Right , quantification of K V 2.1–Ca V 1.2 colocalization volume in WT (black) and NPC1 I1061T (red) neurons in the soma region. n = 47–48 (WT and NPC1 I1061T ) neurons were analyzed across 3 animals. D Diagram detailing HA-TAT-Ca 2+ Channel Association Domain (CCAD) mode of action. E Left , representative super-resolution Airyscan images taken at a focal plane near the PM of CTL (black) and U18 (red) neurons incubated with the CCAD or HA-TAT-Scr scrambled peptide (SCRBL), and co-immunolabeled for Ca V 1.2 and HA. Right , quantification of PM Ca V 1.2 cluster size of CTL (black) and U18-treated (red) neurons co-incubated with CCAD or SCRBL peptide in the soma (left, yellow) and dendrite (right, orange) regions. N = 18 (SCRBL), n = 30 (SCRBL + U18), n = 25 (CCAD) and n = 19 (CCAD + U18) neurons and n = 46 (SCRBL), n = 55 (SCRBL + U18), n = 57 (CCAD) and n = 34 (CCAD + U18) dendrites were analyzed across 2 independent isolations. All error bars represent SEM. Statistical significance was calculated using Mann–Whitney (two-tail) and Unpaired t tests (two-tail) in ( A )–( C ) and two-way ANOVA in ( E ); ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. CTL is control, SCRBL is scramble, U18 is U18666A, and CCAD is calcium channel association domain.

Article Snippet: After 30 min incubation at RT in Tris-Buffered Saline (TBS) buffer supplemented with 0.05% Tween‐20 (TBS-T) and 5% non‐fat dry milk, membranes were exposed O/N at 4 °C to the following primary antibodies: K V 2.1 at 4 μg/mL (NeuroMab, K89/34), Ca V 1.2 at 1:250 (Alomone Labs, acc-003), CDK5 at 1:100 (Santa Cruz Biotechnology, Cat #sc-6247), p35 at 1:100 (Cell Signaling Technology, Cat #C64B10), p39 at 1:100 (Santa Cruz Biotechnology, Cat #sc-365781) or GAPDH at 1:1000 (Proteintech, Cat #10494-I-AP).

Techniques: Immunolabeling, Incubation, MANN-WHITNEY

Journal: Nature Communications

Article Title: NPC1-dependent alterations in K V 2.1–Ca V 1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease

doi: 10.1038/s41467-023-39937-w

Figure Lengend Snippet: A Schematic diagram detailing roscovitine (Rosc) mode of action. B Top , representative super-resolution Airyscan images taken at a focal plane near the PM of CTL (black) or U18-treated (red) neurons co-incubated with roscovitine (Rosc) (cyan) and co-immunolabeled for Ca V 1.2 and K V 2.1. Bottom , quantification of PM KV2.1 and Ca V 1.2 clustering size, and % of the soma occupied by Ca V 1.2–K V 2.1 (left, yellow) and Ca V 1.2–K V 2.1 area in the dendrite region (right, orange) of CTL (black), U18 (red) and Rosc (cyan) neurons. N = 43 (CTL), n = 41 (U18), n = 43 (Rosc) and n = 38 (Rosc+U18) neurons and n = 89 (CTL), n = 94 (U18), n = 82 (Rosc) and n = 71 (Rosc+U18) dendrites were analyzed across 5 independent isolations. All error bars represent SEM. Statistical significance was calculated using a two-way ANOVA test. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. # P < 0.05; ## P < 0.01; #### P < 0.0001. # indicates comparison with the CTL condition. CTL is control, U18 is U18666A, ROSC is Roscovitine.

Article Snippet: After 30 min incubation at RT in Tris-Buffered Saline (TBS) buffer supplemented with 0.05% Tween‐20 (TBS-T) and 5% non‐fat dry milk, membranes were exposed O/N at 4 °C to the following primary antibodies: K V 2.1 at 4 μg/mL (NeuroMab, K89/34), Ca V 1.2 at 1:250 (Alomone Labs, acc-003), CDK5 at 1:100 (Santa Cruz Biotechnology, Cat #sc-6247), p35 at 1:100 (Cell Signaling Technology, Cat #C64B10), p39 at 1:100 (Santa Cruz Biotechnology, Cat #sc-365781) or GAPDH at 1:1000 (Proteintech, Cat #10494-I-AP).

Techniques: Incubation, Immunolabeling

Journal: Nature Communications

Article Title: NPC1-dependent alterations in K V 2.1–Ca V 1.2 nanodomains drive neuronal death in models of Niemann-Pick Type C disease

doi: 10.1038/s41467-023-39937-w

Figure Lengend Snippet: A Schematic diagram of the hypothesis: NPC1 deficient neurons have ER-PM domains enriched in RyR—Ca V 1.2–K v 2.1—SERCA and increased spontaneous Ca 2+ activity at K V 2.1-asociated ER – PM MCSs. B Left , representative super-resolution Airyscan images taken at a focal plane near the PM of CTL (black) and U18-teated (red) neurons co-immunolabeled for Ca V 1.2 and RyR. Right , quantification of % soma occupied by Ca V 1.2–RyR, Ca V 1.2–RyR cluster density and Ca V 1.2–RyR cluster size of CTL (black) and U18-treated (red) neurons in the soma (top, yellow) and dendrite (bottom, orange) region. N = 20–21 (CTL) and n = 26 (U18) neurons and n = 52–53 (CTL) and n = 54–55 (U18) dendrites were analyzed across 2 indepdendent isolations. C Top , schematic diagram of hypothesis: NPC1 deficient neurons (U18, red) have increased Ca 2+ activity and K V 2.1-associated Ca V 1.2, while disrupting K v 2.1–Ca v 1.2 or K V 2.1–VAPA/B interactions (CCAD, purple or FFAT, red-brown) abrogates such effects. Middle left , representative super-resolution TIRF images of neurons transfected with GCamP3-Kv2.1 P4O4W . Bottom left , intensity time series and kymographs of spontaneous activity taken from the square region of interest. Middle right , quantification of GCamP3-Kv2.1 P4O4W peak amplitude of CTL (black), U18 (red), CCAD + U18 (purple) and FFAT + U18 (red-brown) neurons. N = 85 (CTL), n = 141 (U18), n = 23 (CCAD + U18) and n = 10 (FFAT + U18) GCamP3-Kv2.1 P4O4W peaks were analyzed across 3 independent isolations. All error bars represent SEM. Statistical significance was calculated using the following tests: Unpaired (two-tail) and Mann–Whitney t tests (two-tail) in ( B ) and Kruskal–Wallis test in ( C ). ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. # P < 0.05; ## P < 0.01; #### P < 0.0001. # indicates comparison with the CTL condition. CTL is control, SCRBL is scramble, U18 is U18666A, CCAD is calcium channel association domain, and FFAT is two phenylalanines (FF) in an acidic tract.

Article Snippet: After 30 min incubation at RT in Tris-Buffered Saline (TBS) buffer supplemented with 0.05% Tween‐20 (TBS-T) and 5% non‐fat dry milk, membranes were exposed O/N at 4 °C to the following primary antibodies: K V 2.1 at 4 μg/mL (NeuroMab, K89/34), Ca V 1.2 at 1:250 (Alomone Labs, acc-003), CDK5 at 1:100 (Santa Cruz Biotechnology, Cat #sc-6247), p35 at 1:100 (Cell Signaling Technology, Cat #C64B10), p39 at 1:100 (Santa Cruz Biotechnology, Cat #sc-365781) or GAPDH at 1:1000 (Proteintech, Cat #10494-I-AP).

Techniques: Activity Assay, Immunolabeling, Transfection, MANN-WHITNEY

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Ca V 1.2 alternative exon 9* is specifically increased in diabetic heart . ( A ) The membrane expression of Ca V 1.2 α 1C was detected by Western blotting in heart tissues from control and HFD/STZ-treated rats, Na-K ATPase protein was detected as internal control. The relative band densities were analyzed and normalized to Na-K ATPase. n = 6 rats for each group. * P = 0.0166, unpaired t test. ( B ) Schematic diagram shows the locations of the PCR primers designed to amplify and detect rat Ca V 1.2 inclusive of or in the absence of alternative exons in cardiac tissues. Total RNA was extracted from hearts, and PCR products amplified from cDNA libraries were separated on 2.5% agarose gel. Actb mRNA was detected as internal control. Rat Cacna1c mRNAs with exon 8 or 8a were amplified by RT-PCR, followed by digestion with restriction endonuclease BamHI. The value for percent exon 8a inclusion were the lower 2 bands’ intensity divided by the sum of the intensities of upper and lower bands. n = 4 rats for each group. P = 0.4107, unpaired t test. ( C ) The value for percent exon 9* inclusion was the upper band intensity divided by the summed intensities of upper and lower bands. n = 6 rats for each group. **P < 0.0001, unpaired t test. ( D ) The value for percent exon 33 inclusion was also presented as a bar chart. n = 6 rats for each group. P = 0.4107, unpaired t test. ns indicates no significant differences

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Expressing, Western Blot, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Rbfox2 specifically modulates Ca V 1.2 alternative exon 9* splicing in cardiomyocyte . ( A ) H9c2 cells were transfected with nontargeting (NT) or Rbfox2 siRNA for 48 h. The endogenous expression of Rbfox2 protein was detected by Western blotting, the β-actin was detected as internal control. PCR product of Ca V 1.2 E9* channel was amplified from cDNA libraries and separated on 2.5% agarose gel. Actb mRNA was detected as internal control. ( B ) The relative expression of Rbfox2 was normalized to β-actin. ( C ) The value for percent Ca V 1.2 E9* inclusion was the upper band intensity divided by the summed intensities of upper and lower bands, and presented as a bar chart. ( D ) H9c2 cells were transfected with an empty vector, WT Rbfox2, DN Rbfox2 or WT plus DN Rbfox2 expression plasmids, nontreated cells were set as negative control (NC). After 48 h incubation, the expression of Rbfox2 protein was detected by Western blotting, the β-actin was detected as internal control. PCR products were separated on 2–3% agarose gel, which was used to check the proportions of Ca V 1.2 E9* channels. ( E ) Relative expression of Rbfox2 was normalized to β-actin. ( F ) The proportion of Ca V 1.2 E9* channels were analyzed and presented by a bar chart. n = 3 independent experiments. * P < 0.05, **P < 0.01, one-way ANOVA followed by a Tukey’s post hoc test

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Transfection, Expressing, Western Blot, Amplification, Agarose Gel Electrophoresis, Plasmid Preparation, Negative Control, Incubation

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Glycated serum (GS), not D-glucose, decreases Rbfox2 expression but increases Ca V 1.2 E9* channels . ( A ) NRVMs were treated with mannitol or D-glucose, nontreated cells were set as control. After 48 h incubation, the endogenous expression of Rbfox2 protein was detected by Western blotting, the β-actin was detected as internal control. ( B ) Relative Rbfox2 expression was normalized with β-actin in differentially-treated cells. n = 4 independent experiments. P = 0.9854, one-way ANOVA followed by a Tukey’s post hoc test. ( C ) PCR products amplified from cDNA libraries of differentially-treated NRVM were separated on 2.5% agarose gel, and the values of proportion of Ca V 1.2 E9* were analyzed. n = 4 independent experiments. P = 0.9109, one-way ANOVA followed by a Tukey’s post hoc test. ( D ) Serum levels of advanced glycation end-products (AGEs) were measured by an ELISA kit in control (n = 5) and HFD/STZ-treated rats (n = 6). ** P = 0.0042, unpaired t test. ( E ) The levels of AGEs in the lysate of cardiac tissues from different rats’ models (n = 6 each group) were also measured by ELISA. ** P = 0.0027, unpaired t test. ( F ) NRVMs were treated with 10% non-glycated (NG) or glycated serum (GS) for 48 h, after that the cells were harvested for detecting Ca V 1.2 alternative exon 9* and expression of Rbfox2 by RT-PCR and Western blotting, respectively. ( G ) Relative expression of Rbfox2 was also analyzed. n = 3 independent experiments. * P = 0.0478, unpaired t test. ( H ) The values for percent Ca V 1.2 E9* channels were presented. n = 6 independent experiments. P < 0.0001, unpaired t test. ( I - J ) WT and DN Rbfox2 mRNA expression were also determined by real-time RT-PCR in NRVMs treated with NG or GS. Actb mRNA was measured as internal control. n = 5–6 independent experiments. **P < 0.01, unpaired t test. ns indicates no significant differences

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Expressing, Incubation, Western Blot, Amplification, Agarose Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: GS application induces hyperpolarized window currents of Ca V 1.2 channel in NRVMs . ( A ) NRVMs were treated with 10% NG or GS for 48 h, then the membrane protein was extracted. Ca V 1.2 α 1C subunit was detected, and Na-K ATPase was detected as internal control. ( B ) Relative expression of Ca V 1.2 α 1C subunit was normalized with Na-K ATPase. n = 3 independent experiments. P = 0.7251, unpaired t test. ns indicates no significant differences. ( C ) Ca V 1.2 channel currents were recorded from NRVMs with NG, GS or GS + Gö6983 treatment in 10 mmol/L Ba 2+ bath solution. ( D ) I-V relationship curves of Ca V 1.2 channel were recorded under different testing potentials in NRVMs. ( E ) Current densities of Ca V 1.2 channel in NRVMs treated with NG, GS or GS + Gö6983 were presented. ( F ) Plots of steady-state activation (SSA) curve of Ca V 1.2 channel were derived from I - V currents in differentially-treated NRVMs. ( G ) Plots of the steady-state inactivation (SSI) curve were recorded and analyzed in NRVMs. ( H ) Window currents were superimposed from SSI ( f ∞ ) and SSA ( d ∞ ) curves of NG, GS or GS + Gö6983-treated NRVMs

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Expressing, Activation Assay, Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Knockdown of Rbfox2 hyperpolarizes window currents of Ca V 1.2 channel in NRVMs . ( A ) The protein expression of Rbfox2 and β-actin were detected in whole-cell lysate of isolated NRVMs by using Western blotting after transfecting with NT or Rbfox2 siRNAs. The membrane protein was also extracted, and membrane expression of Ca V 1.2 α 1C was checked, Na-K ATPase was detected as a membrane loading control. ( B ) Relative expression level of Rbfox2 was normalized with β-actin in differentially transfected cells, and presented as a bar chart. n = 4 independent experiments. ** P < 0.001, one-way ANOVA followed by a Tukey’s post hoc test. ( C ) Relative Ca V 1.2 α 1C membrane expression was normalized with Na-K ATPase in differentially-transfected cells. n = 4 independent experiments. P = 0.6679, one-way ANOVA followed by a Tukey’s post hoc test. ( D ) Raw traces of Ca V 1.2 whole-cell calcium current recorded from NRVMs treated with NT or Rbfox2 siRNA in 10 mmol/L Ba 2+ external solution. ( E ) I-V relationship of calcium channel current recorded under the different testing potential, increased from − 50 to 50 mV in NRVMs transfected with NT or Rbfox2 siRNA. ( F ) Ca V 1.2 channel current density in NRVMs was analyzed after transfected with NT or Rbfox2 siRNA. ( H ) Plots of steady-state activation (SSA) curve of Ca V 1.2 channel were analyzed from I-V currents in NT or Rbfox2 siRNA-treated NRVMs. ( H ) Plots of the steady-state inactivation (SSI) was also recorded in NRVMs. ( I ) Ca V 1.2 window currents were superimposed from SSI ( f ∞ ) and SSA ( d ∞ ) curves of NRVMs

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Expressing, Isolation, Western Blot, Transfection, Activation Assay

Journal: Cardiovascular Diabetology

Article Title: Dysregulated Rbfox2 produces aberrant splicing of Ca V 1.2 calcium channel in diabetes-induced cardiac hypertrophy

doi: 10.1186/s12933-023-01894-5

Figure Lengend Snippet: Knockdown of Rbfox2 induces cardiomyocyte hypertrophy . ( A ) NRVMs treated with NT or Rbfox2 siRNAs, real-time [Ca 2+ ] i was measured by Ca 2+ fluorescence indicator Fluo-4 AM, Δ[Ca 2+ ] i fluorescence intensities were measured by dividing the changes in the fluorescent signal by the average resting fluorescence. ( B ) Plots of time course of fluorescent intensity after application with NT (n = 20 cells) or Rbfox2 siRNAs (n = 13 cells) in NRVMs from 3 independent experiments, Δ[Ca 2+ ] i was presented as Δ F / F 0 . * P < 0.05, two-way ANOVA followed by Sidak’s multiple comparisons test. ( C ) Representative images of NRVMs transfected with NT or Rbfox2 siRNA, immunofluorescence staining by using anti-a-actinin antibody was applied to determine cell surface area (CSA). ( D ) Analyzed CSA was shown as a bar chart in NT (n = 18 cells) or Rbfox2 siRNA (n = 18 cells) transfected NRVMs from 3 independent experiments. ** P = 0.0005, unpaired t test. ( E ) Rat Nppa, Nppb and Myh7 mRNAs were determined by real-time RT-PCR in differentially-treated NRVMs, rat Actb mRNA was detected as internal control. n = 3–4 independent experiments. * P < 0.05, ** P < 0.01, unpaired t test. ( F ) Illustration of main findings in this study. Under diabetic hyperglycemia, AGEs, not glucose, induces aberrant expression of Rbfox2, which upregulates Ca V 1.2 E9* channels in cardiomyocyte. AGEs/Rbfox2-mediated AS hyperpolarizes cardiac Ca V 1.2 window currents, elevates K + -triggered [Ca 2+ ] i and promotes fetal genes’ transcription, finally induces cardiomyocyte hypertrophy. RAGE, receptor for advanced glycation end-products; RyR2, ryanodine receptor 2; SR, sarcoplasmic reticulum

Article Snippet: Primary antibodies against targeted proteins were used: β-actin (1 mg/mL, HRP-66,009, Proteintech), Rbfox2 (1 mg/mL, NB110-40588, rabbit polyclonal, Novus), Ca V 1.2 α 1C (1.6 µg/mL, ACC-003, rabbit polyclonal, Alomone), Na-K ATPase (1.0 µg/mL, Ab7671, mouse monoclonal, Abcam), RBM20 (0.5 mg/mL, NBP2-27509, goat polyclonal, Novus).

Techniques: Fluorescence, Transfection, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

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: 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