cacna1c  (Alomone Labs)


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    Structured Review

    Alomone Labs cacna1c
    Alternations of L-type calcium channel transcripts, protein, and action potential in KO smooth muscle. (A) PCR validation of alternatively started and spliced exons of <t>Cacna1c</t> in WT and KO jejunum smooth muscle at KO days 5, 10, and 15. NTC is non template control. Primer sets were designed from variant exons in the regions (e.g. E1-3, forward primer spanning a region on exon 1 and reverse primer on exon 3). (B) qPCR data showing decreased expression of Cacna1c variants starting at exon 1 and exon 2 long and short forms (E1-3, E2L-3, and E2L/S-3) at KO days 5, 10, 15, and 20. E1-3, a region spanning exons 1 and 3; E2L-3, a region spanning exon 2 long (L) form and exon 3; E2L/S-3, a region spanning exon 2 long (L) and short (S) forms and exon 3. (C) Western blot showing decreased levels of CACNA1C protein in Srf KO muscle. (D) Consensus sequence of 5’ splice donor and 3’ splice acceptor sites. (E) A topological map of CACNA1C variants. Amino acid sequence is written in small circles. Four motifs are indicated as I-IV and six transmembrane domains, S1-S6. Four pore regions are also indicated. Colors on amino acid sequence show particular regions and domains: red, missing or inserted peptides from differentially spliced exons; purple, voltage sensors in S4 transmembrane domains; green, start codons found in differentially spliced variants (*, start codons deduced from indicated exons that are differentially spliced; blue, β subunit binding domain; brown, CaM (calmodulin) binding domain; orange, PKA (protein kinase A) phosphorylation site. Alignment of alternatively spliced exons E9/10, E24/25, and E32/33 are shown. (F) Isometric force recordings from antrum and colon of WT and KO mice. Bay K8644 (1 μM) and high potassium (K + ) Krebs (36 mM and 72 mM) were applied to the tissues (indicated by bar and arrows). (G) The graph summarizes the results for 9 antral and 5 colonic WT and KO tissues. The responses to Bay K8644, 36 mM K + , and 72 mM K + were significantly decreased in KO antrums, and the responses to 36 mM K + and 72 mM K + were significantly reduced in KO colons compared to WT. * and ** represent p ≤ 0.05 and p ≤ 0.01 respectively.
    Cacna1c, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels"

    Article Title: Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171262

    Alternations of L-type calcium channel transcripts, protein, and action potential in KO smooth muscle. (A) PCR validation of alternatively started and spliced exons of Cacna1c in WT and KO jejunum smooth muscle at KO days 5, 10, and 15. NTC is non template control. Primer sets were designed from variant exons in the regions (e.g. E1-3, forward primer spanning a region on exon 1 and reverse primer on exon 3). (B) qPCR data showing decreased expression of Cacna1c variants starting at exon 1 and exon 2 long and short forms (E1-3, E2L-3, and E2L/S-3) at KO days 5, 10, 15, and 20. E1-3, a region spanning exons 1 and 3; E2L-3, a region spanning exon 2 long (L) form and exon 3; E2L/S-3, a region spanning exon 2 long (L) and short (S) forms and exon 3. (C) Western blot showing decreased levels of CACNA1C protein in Srf KO muscle. (D) Consensus sequence of 5’ splice donor and 3’ splice acceptor sites. (E) A topological map of CACNA1C variants. Amino acid sequence is written in small circles. Four motifs are indicated as I-IV and six transmembrane domains, S1-S6. Four pore regions are also indicated. Colors on amino acid sequence show particular regions and domains: red, missing or inserted peptides from differentially spliced exons; purple, voltage sensors in S4 transmembrane domains; green, start codons found in differentially spliced variants (*, start codons deduced from indicated exons that are differentially spliced; blue, β subunit binding domain; brown, CaM (calmodulin) binding domain; orange, PKA (protein kinase A) phosphorylation site. Alignment of alternatively spliced exons E9/10, E24/25, and E32/33 are shown. (F) Isometric force recordings from antrum and colon of WT and KO mice. Bay K8644 (1 μM) and high potassium (K + ) Krebs (36 mM and 72 mM) were applied to the tissues (indicated by bar and arrows). (G) The graph summarizes the results for 9 antral and 5 colonic WT and KO tissues. The responses to Bay K8644, 36 mM K + , and 72 mM K + were significantly decreased in KO antrums, and the responses to 36 mM K + and 72 mM K + were significantly reduced in KO colons compared to WT. * and ** represent p ≤ 0.05 and p ≤ 0.01 respectively.
    Figure Legend Snippet: Alternations of L-type calcium channel transcripts, protein, and action potential in KO smooth muscle. (A) PCR validation of alternatively started and spliced exons of Cacna1c in WT and KO jejunum smooth muscle at KO days 5, 10, and 15. NTC is non template control. Primer sets were designed from variant exons in the regions (e.g. E1-3, forward primer spanning a region on exon 1 and reverse primer on exon 3). (B) qPCR data showing decreased expression of Cacna1c variants starting at exon 1 and exon 2 long and short forms (E1-3, E2L-3, and E2L/S-3) at KO days 5, 10, 15, and 20. E1-3, a region spanning exons 1 and 3; E2L-3, a region spanning exon 2 long (L) form and exon 3; E2L/S-3, a region spanning exon 2 long (L) and short (S) forms and exon 3. (C) Western blot showing decreased levels of CACNA1C protein in Srf KO muscle. (D) Consensus sequence of 5’ splice donor and 3’ splice acceptor sites. (E) A topological map of CACNA1C variants. Amino acid sequence is written in small circles. Four motifs are indicated as I-IV and six transmembrane domains, S1-S6. Four pore regions are also indicated. Colors on amino acid sequence show particular regions and domains: red, missing or inserted peptides from differentially spliced exons; purple, voltage sensors in S4 transmembrane domains; green, start codons found in differentially spliced variants (*, start codons deduced from indicated exons that are differentially spliced; blue, β subunit binding domain; brown, CaM (calmodulin) binding domain; orange, PKA (protein kinase A) phosphorylation site. Alignment of alternatively spliced exons E9/10, E24/25, and E32/33 are shown. (F) Isometric force recordings from antrum and colon of WT and KO mice. Bay K8644 (1 μM) and high potassium (K + ) Krebs (36 mM and 72 mM) were applied to the tissues (indicated by bar and arrows). (G) The graph summarizes the results for 9 antral and 5 colonic WT and KO tissues. The responses to Bay K8644, 36 mM K + , and 72 mM K + were significantly decreased in KO antrums, and the responses to 36 mM K + and 72 mM K + were significantly reduced in KO colons compared to WT. * and ** represent p ≤ 0.05 and p ≤ 0.01 respectively.

    Techniques Used: Polymerase Chain Reaction, Variant Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Sequencing, Binding Assay, Chick Chorioallantoic Membrane Assay, Mouse Assay

    Model showing the possible molecular pathways by which SRF regulates contractility via DMPK and CACNA1C in SMC.
    Figure Legend Snippet: Model showing the possible molecular pathways by which SRF regulates contractility via DMPK and CACNA1C in SMC.

    Techniques Used:

    Identification of a predominant subtype and alternative transcriptional variants of L-type calcium channels expressed in SMC. (A) Expression levels of L-type calcium channel subtypes in SMC of jejunum and colon. (B) Expression levels of Cacna1c variants in SMC and tissue of jejunum and colon. (C) A genomic map of Cacna1c variants. Five variable regions (V1-5) are indicated. Exons are numbered 1–48. (D) Magnified view of variable regions showing alternatively started or spliced exons (indicated as exon numbers). Seven exons containing alternative transcriptional start sequence are shown by a star (*). Long (L) and short (S) exons that are differentially started or spliced are indicated.
    Figure Legend Snippet: Identification of a predominant subtype and alternative transcriptional variants of L-type calcium channels expressed in SMC. (A) Expression levels of L-type calcium channel subtypes in SMC of jejunum and colon. (B) Expression levels of Cacna1c variants in SMC and tissue of jejunum and colon. (C) A genomic map of Cacna1c variants. Five variable regions (V1-5) are indicated. Exons are numbered 1–48. (D) Magnified view of variable regions showing alternatively started or spliced exons (indicated as exon numbers). Seven exons containing alternative transcriptional start sequence are shown by a star (*). Long (L) and short (S) exons that are differentially started or spliced are indicated.

    Techniques Used: Expressing, Sequencing

    2) Product Images from "Ca2+/Calmodulin-dependent Protein Kinase II-dependent Remodeling of Ca2+ Current in Pressure Overload Heart Failure *"

    Article Title: Ca2+/Calmodulin-dependent Protein Kinase II-dependent Remodeling of Ca2+ Current in Pressure Overload Heart Failure *

    Journal:

    doi: 10.1074/jbc.M803043200

    A , surface expression of α1c protein in SEN and SEP myocytes from sham and HF LV lysates. Surface proteins were biotinylated and immobilized on avidin agarose, and equal amounts of protein were subjected to SDS-PAGE and Western blot. Densitometric
    Figure Legend Snippet: A , surface expression of α1c protein in SEN and SEP myocytes from sham and HF LV lysates. Surface proteins were biotinylated and immobilized on avidin agarose, and equal amounts of protein were subjected to SDS-PAGE and Western blot. Densitometric

    Techniques Used: Expressing, Avidin-Biotin Assay, SDS Page, Western Blot

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    Alomone Labs acc 003 antibody
    Location of antibody epitopes. Shown is a schematic of the Ca v 1.2 α 1 1.2 subunit, in which regions used as immunogens for the depicted antibodies are identified by arrows. Exact residues are listed in the table and numbered according to α 1 1.2 given in Gene Bank Accession number CAA33546. FP1, CNC1, and <t>ACC-003</t> are directed against the loop between domains II and III, pS1700 against phosphorylated S1700, pS1928 against phosphorylated S1928, and CNC2 against residues 2122-2138 of α 1 1.2, which are ~40 residues upstream of the very C terminus of α 1 1.2.
    Acc 003 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Alomone Labs anti cav1 2 rabbit polyclonal
    <t>CaV1.2</t> downregulation mediates impairment of the first phase of insulin secretion in Tardbp knockdown MIN6 cells. ( A ) mRNA expression levels of Cacna1c and Cacna2d1 that encode CaV1.2 and Ca2d1, respectively (control siRNA, Control; Tardbp siRNA, T1 or T2) ( Cacna1c , n = 6; Cacna2d1 , n = 12 each, 1-way ANOVA). ( B ) Immunoblotting of MIN6 cells transfected with control, T1, or T2 (n = 6 each, one-way ANOVA). ( C ) Mock or CaV1.2 plasmid was cotransfected into MIN6 cells with control or T1 siRNA ( n = 4 each, 1-way ANOVA). ( D ) The insulin assay with low and high glucose ( n = 6 each, 1-way ANOVA). ( E ) In situ hybridization of the islets of disease control and ALS subjects with a human CACNA1C mRNA antisense probe. Scale bars: 20 μm. ( F ) Quantitative analysis of in situ hybridization of human CACNA1C , CACNA1D , and CACNA1A ( n = 4 patients each, unpaired t test). Values are mean ± SEM. * P
    Anti Cav1 2 Rabbit Polyclonal, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Location of antibody epitopes. Shown is a schematic of the Ca v 1.2 α 1 1.2 subunit, in which regions used as immunogens for the depicted antibodies are identified by arrows. Exact residues are listed in the table and numbered according to α 1 1.2 given in Gene Bank Accession number CAA33546. FP1, CNC1, and ACC-003 are directed against the loop between domains II and III, pS1700 against phosphorylated S1700, pS1928 against phosphorylated S1928, and CNC2 against residues 2122-2138 of α 1 1.2, which are ~40 residues upstream of the very C terminus of α 1 1.2.

    Journal: F1000Research

    Article Title: Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

    doi: 10.12688/f1000research.11808.1

    Figure Lengend Snippet: Location of antibody epitopes. Shown is a schematic of the Ca v 1.2 α 1 1.2 subunit, in which regions used as immunogens for the depicted antibodies are identified by arrows. Exact residues are listed in the table and numbered according to α 1 1.2 given in Gene Bank Accession number CAA33546. FP1, CNC1, and ACC-003 are directed against the loop between domains II and III, pS1700 against phosphorylated S1700, pS1928 against phosphorylated S1928, and CNC2 against residues 2122-2138 of α 1 1.2, which are ~40 residues upstream of the very C terminus of α 1 1.2.

    Article Snippet: Concordant with this idea, neither the ACC-003 antibody that we received from Alomone Labs that recognized a 130 kDa band in WT and cKO brains nor our CNC1 antibody recognized the strong 150 kDa band seen with FP1 in brain lysate.

    Techniques:

    Differential recognition of the strong 150 kDa FP1 band in lysate and weak 150 kDa band by FP1, CNC1, and ACC-003 after IP of α 1 1.2 with FP1. Immunoblots with CNC1 ( A , B ), FP1 ( C ), and ACC-003 ( D , E ) of Triton X-100 extracts from WT mice (lysate) and after immunoprecipitation with FP1 from cKO and WT mice. Gels were polymerized from 8% acrylamide. Note that a weak 150 kDa band is detected by CNC1, FP1, and ACC-003 after enrichment of α 1 1.2 by immunoprecipitation with FP1 but the strongly immunoreactive 150 kDa band detected by FP1 in lysate is not detectable by either CNC1 or ACC-003.

    Journal: F1000Research

    Article Title: Proteolytic processing of the L-type Ca2+ channel alpha11.2 subunit in neurons

    doi: 10.12688/f1000research.11808.1

    Figure Lengend Snippet: Differential recognition of the strong 150 kDa FP1 band in lysate and weak 150 kDa band by FP1, CNC1, and ACC-003 after IP of α 1 1.2 with FP1. Immunoblots with CNC1 ( A , B ), FP1 ( C ), and ACC-003 ( D , E ) of Triton X-100 extracts from WT mice (lysate) and after immunoprecipitation with FP1 from cKO and WT mice. Gels were polymerized from 8% acrylamide. Note that a weak 150 kDa band is detected by CNC1, FP1, and ACC-003 after enrichment of α 1 1.2 by immunoprecipitation with FP1 but the strongly immunoreactive 150 kDa band detected by FP1 in lysate is not detectable by either CNC1 or ACC-003.

    Article Snippet: Concordant with this idea, neither the ACC-003 antibody that we received from Alomone Labs that recognized a 130 kDa band in WT and cKO brains nor our CNC1 antibody recognized the strong 150 kDa band seen with FP1 in brain lysate.

    Techniques: Western Blot, Mouse Assay, Immunoprecipitation

    Effect of CAL on I Ca , LTCC expression and the systolic Ca transient. A: Representative L-type Ca currents (I Ca ) recorded at 0 mV from Sham (left) and CAL (right) myocytes. B: Mean (± SEM) I Ca density-voltage relations from Sham (circles, n = 50/15) and CAL (squares, n = 41/15) myocytes. C: Representative Western blots of Sham (S) and CAL lysates probed with antibodies against LTCC (top) or GAPDH (bottom). D: Representative whole-cell Ca transients from Sham (grey line) and CAL (black line) myocytes during field stimulation (left); the right panel shows the early rise on an expanded time scale. E: Mean (± SEM) peak ΔF/F 0 , time to peak (TTP), and rate of rise (ΔF/F 0 .ms − 1 ) of the Ca transient in Sham (open bars, n = 10) and CAL (filled bars, n = 16) myocytes. ⁎⁎⁎ p

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts

    doi: 10.1016/j.yjmcc.2015.06.012

    Figure Lengend Snippet: Effect of CAL on I Ca , LTCC expression and the systolic Ca transient. A: Representative L-type Ca currents (I Ca ) recorded at 0 mV from Sham (left) and CAL (right) myocytes. B: Mean (± SEM) I Ca density-voltage relations from Sham (circles, n = 50/15) and CAL (squares, n = 41/15) myocytes. C: Representative Western blots of Sham (S) and CAL lysates probed with antibodies against LTCC (top) or GAPDH (bottom). D: Representative whole-cell Ca transients from Sham (grey line) and CAL (black line) myocytes during field stimulation (left); the right panel shows the early rise on an expanded time scale. E: Mean (± SEM) peak ΔF/F 0 , time to peak (TTP), and rate of rise (ΔF/F 0 .ms − 1 ) of the Ca transient in Sham (open bars, n = 10) and CAL (filled bars, n = 16) myocytes. ⁎⁎⁎ p

    Article Snippet: The blot was probed with anti-LTCC antibody (ACC-003; Alomone, Israel) or anti-GAPDH (G9545; Sigma) and protein bands visualized using relevant peroxidase-conjugated secondary antibodies, chemiluminescence, and autoradiography.

    Techniques: Expressing, Western Blot

    Co-immunoprecipitation shows a biochemical association between KCHIP1 and KV4.2/4.3. ( a , b ) Tissue samples from adult rat heart were lysed and immunoprecipitated (IP) with anti-KCHIP1. The membranes were then immunoblotted (IB) using anti-KV4.2/4.3 ( a ) and anti-Cav1.2 ( b ). Beads conjugated with IgG isotype were used as negative control (NC). There is a biochemical association between KCHIP1 and KV4.2/4.3 ( a ), but no association between KCHIP1 and Cav1.2 ( b ). Data are representative of three independent experiments. Full-length blots are presented in Supplementary Fig. 6 .

    Journal: Nature Communications

    Article Title: Genome-wide screening identifies a KCNIP1 copy number variant as a genetic predictor for atrial fibrillation

    doi: 10.1038/ncomms10190

    Figure Lengend Snippet: Co-immunoprecipitation shows a biochemical association between KCHIP1 and KV4.2/4.3. ( a , b ) Tissue samples from adult rat heart were lysed and immunoprecipitated (IP) with anti-KCHIP1. The membranes were then immunoblotted (IB) using anti-KV4.2/4.3 ( a ) and anti-Cav1.2 ( b ). Beads conjugated with IgG isotype were used as negative control (NC). There is a biochemical association between KCHIP1 and KV4.2/4.3 ( a ), but no association between KCHIP1 and Cav1.2 ( b ). Data are representative of three independent experiments. Full-length blots are presented in Supplementary Fig. 6 .

    Article Snippet: The samples were then subjected to SDS–polyacrylamide gel electrophoresis and immunoblotted with an anti-Kv4.2/4.3 (1:500; Santa Cruz) or anti-Cav1.2 (1:5,000; Alomone Labs) antibodies.

    Techniques: Immunoprecipitation, Negative Control

    CaV1.2 downregulation mediates impairment of the first phase of insulin secretion in Tardbp knockdown MIN6 cells. ( A ) mRNA expression levels of Cacna1c and Cacna2d1 that encode CaV1.2 and Ca2d1, respectively (control siRNA, Control; Tardbp siRNA, T1 or T2) ( Cacna1c , n = 6; Cacna2d1 , n = 12 each, 1-way ANOVA). ( B ) Immunoblotting of MIN6 cells transfected with control, T1, or T2 (n = 6 each, one-way ANOVA). ( C ) Mock or CaV1.2 plasmid was cotransfected into MIN6 cells with control or T1 siRNA ( n = 4 each, 1-way ANOVA). ( D ) The insulin assay with low and high glucose ( n = 6 each, 1-way ANOVA). ( E ) In situ hybridization of the islets of disease control and ALS subjects with a human CACNA1C mRNA antisense probe. Scale bars: 20 μm. ( F ) Quantitative analysis of in situ hybridization of human CACNA1C , CACNA1D , and CACNA1A ( n = 4 patients each, unpaired t test). Values are mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets

    doi: 10.1172/JCI124481

    Figure Lengend Snippet: CaV1.2 downregulation mediates impairment of the first phase of insulin secretion in Tardbp knockdown MIN6 cells. ( A ) mRNA expression levels of Cacna1c and Cacna2d1 that encode CaV1.2 and Ca2d1, respectively (control siRNA, Control; Tardbp siRNA, T1 or T2) ( Cacna1c , n = 6; Cacna2d1 , n = 12 each, 1-way ANOVA). ( B ) Immunoblotting of MIN6 cells transfected with control, T1, or T2 (n = 6 each, one-way ANOVA). ( C ) Mock or CaV1.2 plasmid was cotransfected into MIN6 cells with control or T1 siRNA ( n = 4 each, 1-way ANOVA). ( D ) The insulin assay with low and high glucose ( n = 6 each, 1-way ANOVA). ( E ) In situ hybridization of the islets of disease control and ALS subjects with a human CACNA1C mRNA antisense probe. Scale bars: 20 μm. ( F ) Quantitative analysis of in situ hybridization of human CACNA1C , CACNA1D , and CACNA1A ( n = 4 patients each, unpaired t test). Values are mean ± SEM. * P

    Article Snippet: After denaturation, each cell lysate was separated by SDS-PAGE (5%–20% gradient gel) and analyzed by immunoblotting with ECL Plus detection reagents (NEL104001EA, PerkinElmer) using the following primary antibodies: anti–TDP-43 rabbit polyclonal (1:3000; 10782-2-AP, Proteintech), GAPDH mouse monoclonal (1:1000; MBL International Corporation), and anti-CaV1.2 rabbit polyclonal (1:2000; ACC-003, Alomone Labs).

    Techniques: Expressing, Transfection, Plasmid Preparation, In Situ Hybridization

    Loss of TDP-43 reduces insulin secretion by downregulating CaV1.2 calcium channel expression in MIN6 cells.

    Journal: The Journal of Clinical Investigation

    Article Title: TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets

    doi: 10.1172/JCI124481

    Figure Lengend Snippet: Loss of TDP-43 reduces insulin secretion by downregulating CaV1.2 calcium channel expression in MIN6 cells.

    Article Snippet: After denaturation, each cell lysate was separated by SDS-PAGE (5%–20% gradient gel) and analyzed by immunoblotting with ECL Plus detection reagents (NEL104001EA, PerkinElmer) using the following primary antibodies: anti–TDP-43 rabbit polyclonal (1:3000; 10782-2-AP, Proteintech), GAPDH mouse monoclonal (1:1000; MBL International Corporation), and anti-CaV1.2 rabbit polyclonal (1:2000; ACC-003, Alomone Labs).

    Techniques: Expressing

    TDP-43 regulates the transcription of CaV1.2 calcium channels. ( A ) Immunoprecipitation (IP) of lysates from MIN6 cells overexpressing V5-tagged TDP-43 with rabbit IgG or an anti-V5 antibody (IP-V5), analyzed with immunoblotting with the indicated antibody. ( B ) RNA-IP of V5 in cultured MIN6 cells. Bound RNA was analyzed with qRT-PCR using the indicated primers. IP efficiency was calculated relative to input. Representative data from triplicate experiments are shown. ( C ) Schematics of mature mRNA (exon), premature mRNA (intron), and putative promoter are shown over the Cacna1c gene. ( D ) Each mature mRNA (exon) were analyzed with qRT-PCR using the indicated primers ( n = 6 each, unpaired t test). ( E ) mRNA stability was measured in MIN6 cells (control, T1) treated with 10 mg/mL actinomycin D with qRT-PCR using the indicated Cacna1c and Tuba1a primers ( n = 4 each, 2-way ANOVA). The mRNA levels relative to pretreatment were plotted against time after treatment. Residual mRNA levels were compared at 2, 4, 8, and 12 hours after treatment. ( F ) Each premature mRNA (intron) were analyzed with qRT-PCR using the indicated primers ( n = 6 each, unpaired t test). ( G ) Luciferase reporter assay of the –1542/+257 mouse Cacna1c promoter ( n = 10 each, unpaired t test). ( H ) Human CACNA1C promoter luciferase reporter assay using the LightSwitch promoter reporter GoClone collection ( n = 11 each, unpaired t test). Values are mean ± SEM. * P

    Journal: The Journal of Clinical Investigation

    Article Title: TDP-43 regulates early-phase insulin secretion via CaV1.2-mediated exocytosis in islets

    doi: 10.1172/JCI124481

    Figure Lengend Snippet: TDP-43 regulates the transcription of CaV1.2 calcium channels. ( A ) Immunoprecipitation (IP) of lysates from MIN6 cells overexpressing V5-tagged TDP-43 with rabbit IgG or an anti-V5 antibody (IP-V5), analyzed with immunoblotting with the indicated antibody. ( B ) RNA-IP of V5 in cultured MIN6 cells. Bound RNA was analyzed with qRT-PCR using the indicated primers. IP efficiency was calculated relative to input. Representative data from triplicate experiments are shown. ( C ) Schematics of mature mRNA (exon), premature mRNA (intron), and putative promoter are shown over the Cacna1c gene. ( D ) Each mature mRNA (exon) were analyzed with qRT-PCR using the indicated primers ( n = 6 each, unpaired t test). ( E ) mRNA stability was measured in MIN6 cells (control, T1) treated with 10 mg/mL actinomycin D with qRT-PCR using the indicated Cacna1c and Tuba1a primers ( n = 4 each, 2-way ANOVA). The mRNA levels relative to pretreatment were plotted against time after treatment. Residual mRNA levels were compared at 2, 4, 8, and 12 hours after treatment. ( F ) Each premature mRNA (intron) were analyzed with qRT-PCR using the indicated primers ( n = 6 each, unpaired t test). ( G ) Luciferase reporter assay of the –1542/+257 mouse Cacna1c promoter ( n = 10 each, unpaired t test). ( H ) Human CACNA1C promoter luciferase reporter assay using the LightSwitch promoter reporter GoClone collection ( n = 11 each, unpaired t test). Values are mean ± SEM. * P

    Article Snippet: After denaturation, each cell lysate was separated by SDS-PAGE (5%–20% gradient gel) and analyzed by immunoblotting with ECL Plus detection reagents (NEL104001EA, PerkinElmer) using the following primary antibodies: anti–TDP-43 rabbit polyclonal (1:3000; 10782-2-AP, Proteintech), GAPDH mouse monoclonal (1:1000; MBL International Corporation), and anti-CaV1.2 rabbit polyclonal (1:2000; ACC-003, Alomone Labs).

    Techniques: Immunoprecipitation, Cell Culture, Quantitative RT-PCR, Luciferase, Reporter Assay