anti ca v 1 3 polyclonal rabbit antibody  (Alomone Labs)


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    Alomone Labs anti ca v 1 3 polyclonal rabbit antibody
    a Confocal images showing an overlay of Halo-Ca V 1.3 (JF646-HTL, magenta) with either PM (green) or F-actin signals (green) generated by three-color co-staining. b Orthogonal XZ image of a cell expressing Halo-Ca V 1.3 (magenta) and GFP- Rab 11a (green). Two principal imaging planes used for colocalization analysis are highlighted by white boxes: Medial cell sectioning (1) used for protein localization in ( a , c ), basal plane imaging (2) used for quantitative colocalization analysis in ( d ). c Confocal images of cells co-expressing Halo-Ca V 1.3 with GFP- Rab 4a, GFP- Rab 5a, and GFP- Rab 11a, respectively. d Representative segmentation maps of cell surface localized Halo-Ca V 1.3 signals (magenta, STED) together with each indicated endosomal marker (green, confocal). Colocalization is shown in black. Scale bars: 5 µm ( a – c ), 1 µm ( d ).
    Anti Ca V 1 3 Polyclonal Rabbit Antibody, 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
    https://www.bioz.com/result/anti ca v 1 3 polyclonal rabbit antibody/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ca v 1 3 polyclonal rabbit antibody - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy"

    Article Title: Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

    Journal: Communications Biology

    doi: 10.1038/s42003-024-06313-3

    a Confocal images showing an overlay of Halo-Ca V 1.3 (JF646-HTL, magenta) with either PM (green) or F-actin signals (green) generated by three-color co-staining. b Orthogonal XZ image of a cell expressing Halo-Ca V 1.3 (magenta) and GFP- Rab 11a (green). Two principal imaging planes used for colocalization analysis are highlighted by white boxes: Medial cell sectioning (1) used for protein localization in ( a , c ), basal plane imaging (2) used for quantitative colocalization analysis in ( d ). c Confocal images of cells co-expressing Halo-Ca V 1.3 with GFP- Rab 4a, GFP- Rab 5a, and GFP- Rab 11a, respectively. d Representative segmentation maps of cell surface localized Halo-Ca V 1.3 signals (magenta, STED) together with each indicated endosomal marker (green, confocal). Colocalization is shown in black. Scale bars: 5 µm ( a – c ), 1 µm ( d ).
    Figure Legend Snippet: a Confocal images showing an overlay of Halo-Ca V 1.3 (JF646-HTL, magenta) with either PM (green) or F-actin signals (green) generated by three-color co-staining. b Orthogonal XZ image of a cell expressing Halo-Ca V 1.3 (magenta) and GFP- Rab 11a (green). Two principal imaging planes used for colocalization analysis are highlighted by white boxes: Medial cell sectioning (1) used for protein localization in ( a , c ), basal plane imaging (2) used for quantitative colocalization analysis in ( d ). c Confocal images of cells co-expressing Halo-Ca V 1.3 with GFP- Rab 4a, GFP- Rab 5a, and GFP- Rab 11a, respectively. d Representative segmentation maps of cell surface localized Halo-Ca V 1.3 signals (magenta, STED) together with each indicated endosomal marker (green, confocal). Colocalization is shown in black. Scale bars: 5 µm ( a – c ), 1 µm ( d ).

    Techniques Used: Generated, Staining, Expressing, Imaging, Marker

    a Live-cell confocal images of transiently transfected HEK293 cells show cell-surface localized signals of Halo-Ca V 1.3 channels labeled with JF646-HTL (‘Fire’ LUT). The lower-hand image demonstrates high signal densities in the basal PM imaging plane. b Cell-surface STED nanoscopy revealed a clustered distribution of Halo-Ca V 1.3 signals, as resolved by STED but not by confocal imaging. Individual cluster signals were segmented as shown by white outlines. For each cluster, the signal brightness was corrected for the local background prior to molecular counting by brightness referencing. c Frequency distribution of the cluster area obtained from segmented signals ( n clusters = 9459 clusters from n cell = 75). The values in blue provide the corresponding diameter of the clusters assuming a round cluster shape. The figure legend shows the mean and s.d. for the cluster area and diameter. d Frequency distribution of fluorescent channel counts within the segmented cluster signals as determined by brightness referencing. e Scatter graph showing the relationship between fluorescent channel counts and cluster area. The correlation was quantified by Spearman’s r = 0.87 ( p < 0.0001). f Scatter graph of the molecular density and cluster area with r = 0.18 ( p < 0.0001). Scale bars: 20 µm ( a ), 1 µm ( b ).
    Figure Legend Snippet: a Live-cell confocal images of transiently transfected HEK293 cells show cell-surface localized signals of Halo-Ca V 1.3 channels labeled with JF646-HTL (‘Fire’ LUT). The lower-hand image demonstrates high signal densities in the basal PM imaging plane. b Cell-surface STED nanoscopy revealed a clustered distribution of Halo-Ca V 1.3 signals, as resolved by STED but not by confocal imaging. Individual cluster signals were segmented as shown by white outlines. For each cluster, the signal brightness was corrected for the local background prior to molecular counting by brightness referencing. c Frequency distribution of the cluster area obtained from segmented signals ( n clusters = 9459 clusters from n cell = 75). The values in blue provide the corresponding diameter of the clusters assuming a round cluster shape. The figure legend shows the mean and s.d. for the cluster area and diameter. d Frequency distribution of fluorescent channel counts within the segmented cluster signals as determined by brightness referencing. e Scatter graph showing the relationship between fluorescent channel counts and cluster area. The correlation was quantified by Spearman’s r = 0.87 ( p < 0.0001). f Scatter graph of the molecular density and cluster area with r = 0.18 ( p < 0.0001). Scale bars: 20 µm ( a ), 1 µm ( b ).

    Techniques Used: Transfection, Labeling, Imaging

    a Schematics of the production of GPMVs derived from cells and the spreading of GPMVs on a solid support. b Confocal image of the Halo-Ca V 1.3 signal (‘Fire’ LUT) showing a GPMV attached to a HEK293 cell. c Upright STED image resolving spot-like fluorescent Halo-Ca V 1.3 clusters in the detached GPMV. d Upright STED image of a supported plasma membrane bilayer (SPMB) obtained by spreading a GPMV on soda lime-glass (left image: R18, red; right image: Halo-Ca V 1.3, magenta). e Distribution of the SPMB surface areas with a mean area of 410 ± 340 µm² (mean ± s.d.) ( n SPMB = 232). Scale bars: 5 µm ( b ), 10 µm ( c , d ).
    Figure Legend Snippet: a Schematics of the production of GPMVs derived from cells and the spreading of GPMVs on a solid support. b Confocal image of the Halo-Ca V 1.3 signal (‘Fire’ LUT) showing a GPMV attached to a HEK293 cell. c Upright STED image resolving spot-like fluorescent Halo-Ca V 1.3 clusters in the detached GPMV. d Upright STED image of a supported plasma membrane bilayer (SPMB) obtained by spreading a GPMV on soda lime-glass (left image: R18, red; right image: Halo-Ca V 1.3, magenta). e Distribution of the SPMB surface areas with a mean area of 410 ± 340 µm² (mean ± s.d.) ( n SPMB = 232). Scale bars: 5 µm ( b ), 10 µm ( c , d ).

    Techniques Used: Derivative Assay, Membrane

    a SPMB (R18, red) obtained upon spreading a GPMV on borosilicate glass (left image, confocal). The SPMB contains Halo-Ca V 1.3 channel clusters (‘Fire’ LUT) (right image). The bottom part shows the confocal image, and the top part is the STED image. b Comparison of the appearance of the Halo-Ca V 1.3 clusters in SPMBs and in living cells. Single Halo-Ca V 1.3 clusters are resolved, allowing us to determine the c cluster area, d the channel counts, and e the molecular density (box: IQR, dot: mean, line: med.; whiskers: 5–95%) [Mann–Whitney U -test: **** P ≤ 0.0001]. n is the number of analyzed clusters. f , g Two-dimensional kernel density (2D-KDF) of Ca V 1.3 cluster properties of cells (blue) and SPMBs (black). f 2D-KDF of the channel counts vs. cluster area and g 2D-KDF of the molecular density vs. cluster area ( n cell = 75, n SPMB = 20). Scale bars: 10 µm ( a ); 500 nm ( b ).
    Figure Legend Snippet: a SPMB (R18, red) obtained upon spreading a GPMV on borosilicate glass (left image, confocal). The SPMB contains Halo-Ca V 1.3 channel clusters (‘Fire’ LUT) (right image). The bottom part shows the confocal image, and the top part is the STED image. b Comparison of the appearance of the Halo-Ca V 1.3 clusters in SPMBs and in living cells. Single Halo-Ca V 1.3 clusters are resolved, allowing us to determine the c cluster area, d the channel counts, and e the molecular density (box: IQR, dot: mean, line: med.; whiskers: 5–95%) [Mann–Whitney U -test: **** P ≤ 0.0001]. n is the number of analyzed clusters. f , g Two-dimensional kernel density (2D-KDF) of Ca V 1.3 cluster properties of cells (blue) and SPMBs (black). f 2D-KDF of the channel counts vs. cluster area and g 2D-KDF of the molecular density vs. cluster area ( n cell = 75, n SPMB = 20). Scale bars: 10 µm ( a ); 500 nm ( b ).

    Techniques Used: Comparison, MANN-WHITNEY

    a SPMB with Halo-Ca V 1.3 clusters (labeled with JF646-HTL, ‘Fire’ LUT, brightness scale identical for both images) without (no ISO) and with (ISO) treatment. b Cluster density as a function of the channel counts (mean ± s.e.m.), (untreated SPMBs, black; ISO-treated SPMBs, red). c Two-dimensional kernel densities (2D-KDF) of the channel counts as a function of cluster area for untreated SPMBs (black) and ISO-treated SPMBs (red). d 2D-KDF of the molecular density vs. cluster area for untreated (no ISO, black) and ISO-treated (ISO, red) SPMBs. Reproduced in four experiments with n SPMB,no ISO = 20, n clusters,no ISO = 9862; n SPMB,ISO = 17, n clusters,ISO = 5818. Scale bar: 1 µm ( a ).
    Figure Legend Snippet: a SPMB with Halo-Ca V 1.3 clusters (labeled with JF646-HTL, ‘Fire’ LUT, brightness scale identical for both images) without (no ISO) and with (ISO) treatment. b Cluster density as a function of the channel counts (mean ± s.e.m.), (untreated SPMBs, black; ISO-treated SPMBs, red). c Two-dimensional kernel densities (2D-KDF) of the channel counts as a function of cluster area for untreated SPMBs (black) and ISO-treated SPMBs (red). d 2D-KDF of the molecular density vs. cluster area for untreated (no ISO, black) and ISO-treated (ISO, red) SPMBs. Reproduced in four experiments with n SPMB,no ISO = 20, n clusters,no ISO = 9862; n SPMB,ISO = 17, n clusters,ISO = 5818. Scale bar: 1 µm ( a ).

    Techniques Used: Labeling

    polyclonal rabbit anti trpc3 antibody  (Alomone Labs)


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    Alomone Labs polyclonal rabbit anti trpc3 antibody
    L687, a <t>TRPC3/C6/C7</t> activator, induces intracellular Ca 2+ uptake. ( A ) Analysis of intracellular Ca 2+ influx with various concentrations of L687 or PPZ2 in HEK293 cells, overexpressing TRPC3/C6/C7, respectively, in the presence of 2 mM Ca 2+ . Ca 2+ influx was recorded following the addition of each compound. ( B ) Analysis of Ca 2+ influx into the A549 cells in the presence of 2 mM Ca 2+ . After adding PPZ2 (30 μM) or L687 (3, 10 and 30 μM), the Ca 2+ influx was analysed. TRPC, transient receptor potential canonical.
    Polyclonal Rabbit Anti Trpc3 Antibody, 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
    https://www.bioz.com/result/polyclonal rabbit anti trpc3 antibody/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit anti trpc3 antibody - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides"

    Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkae245

    L687, a TRPC3/C6/C7 activator, induces intracellular Ca 2+ uptake. ( A ) Analysis of intracellular Ca 2+ influx with various concentrations of L687 or PPZ2 in HEK293 cells, overexpressing TRPC3/C6/C7, respectively, in the presence of 2 mM Ca 2+ . Ca 2+ influx was recorded following the addition of each compound. ( B ) Analysis of Ca 2+ influx into the A549 cells in the presence of 2 mM Ca 2+ . After adding PPZ2 (30 μM) or L687 (3, 10 and 30 μM), the Ca 2+ influx was analysed. TRPC, transient receptor potential canonical.
    Figure Legend Snippet: L687, a TRPC3/C6/C7 activator, induces intracellular Ca 2+ uptake. ( A ) Analysis of intracellular Ca 2+ influx with various concentrations of L687 or PPZ2 in HEK293 cells, overexpressing TRPC3/C6/C7, respectively, in the presence of 2 mM Ca 2+ . Ca 2+ influx was recorded following the addition of each compound. ( B ) Analysis of Ca 2+ influx into the A549 cells in the presence of 2 mM Ca 2+ . After adding PPZ2 (30 μM) or L687 (3, 10 and 30 μM), the Ca 2+ influx was analysed. TRPC, transient receptor potential canonical.

    Techniques Used:

    Effects of the TRPC3/C6/C7 activator L687 or CBD on ASO activity. ( A ) Analysis of MALAT1 expression by adding MALAT1_ASO with L687. MALAT1_ASO (100 nM) was added to either 1–30 μM L687 in the medium. A549 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of REST versus GAPDH was compared with that of MALAT1 in untreated A549 cells. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of mock DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) Analysis of SRRM4 expression by adding SRRM4_ASO with L687. SRRM4_ASO (100 nM) was added with 30 μM L687 to the medium. N417 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of SRRM4 versus GAPDH was compared with that of SRRM4 in untreated N417 cells. ( C , D ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (100 nM) was added to 10 and 30 μM of L687 or CBD in the medium. A549 cells were collected after 48 h, and the relative expression of REST versus GAPDH was analysed. ( E ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (20 and 100 nM) was added to 30 μM L687 in the medium. A549 cells were collected after 72 h, and the relative expression of REST versus GAPDH was analysed. ( F ) Analysis of ERBB2 expression by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. SK-OV-3 cells were collected after 48 h, and the relative expression of ERBB2 versus β-actin was analysed. ( G ) Viability analysis of SK-OV-3 by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. Cell viability was analysed after 48 h of incubation. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative PCR; TRPC, transient receptor potential canonical.
    Figure Legend Snippet: Effects of the TRPC3/C6/C7 activator L687 or CBD on ASO activity. ( A ) Analysis of MALAT1 expression by adding MALAT1_ASO with L687. MALAT1_ASO (100 nM) was added to either 1–30 μM L687 in the medium. A549 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of REST versus GAPDH was compared with that of MALAT1 in untreated A549 cells. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of mock DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) Analysis of SRRM4 expression by adding SRRM4_ASO with L687. SRRM4_ASO (100 nM) was added with 30 μM L687 to the medium. N417 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of SRRM4 versus GAPDH was compared with that of SRRM4 in untreated N417 cells. ( C , D ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (100 nM) was added to 10 and 30 μM of L687 or CBD in the medium. A549 cells were collected after 48 h, and the relative expression of REST versus GAPDH was analysed. ( E ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (20 and 100 nM) was added to 30 μM L687 in the medium. A549 cells were collected after 72 h, and the relative expression of REST versus GAPDH was analysed. ( F ) Analysis of ERBB2 expression by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. SK-OV-3 cells were collected after 48 h, and the relative expression of ERBB2 versus β-actin was analysed. ( G ) Viability analysis of SK-OV-3 by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. Cell viability was analysed after 48 h of incubation. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative PCR; TRPC, transient receptor potential canonical.

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction

    Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.
    Figure Legend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

    Techniques Used: Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy

    primary polyclonal rabbit anti trpc6 antibody  (Alomone Labs)


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    Alomone Labs primary polyclonal rabbit anti trpc6 antibody
    Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or <t>TRPC6</t> siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.
    Primary Polyclonal Rabbit Anti Trpc6 Antibody, 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
    https://www.bioz.com/result/primary polyclonal rabbit anti trpc6 antibody/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    primary polyclonal rabbit anti trpc6 antibody - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides"

    Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkae245

    Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.
    Figure Legend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

    Techniques Used: Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy

    aqp4 primary rabbit polyclonal antibody  (Alomone Labs)


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    Alomone Labs aqp4 primary rabbit polyclonal antibody
    Tear secretion of WT and <t>AQP4</t> KO mice. Data from both eyes of four mice (2 males and 2 females) were averaged and are presented as means ± SEM. Tear secretion of WT and AQP4 KO animals did not differ significantly.
    Aqp4 Primary Rabbit Polyclonal Antibody, 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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    Images

    1) Product Images from "The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice"

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.65.5.30

    Tear secretion of WT and AQP4 KO mice. Data from both eyes of four mice (2 males and 2 females) were averaged and are presented as means ± SEM. Tear secretion of WT and AQP4 KO animals did not differ significantly.
    Figure Legend Snippet: Tear secretion of WT and AQP4 KO mice. Data from both eyes of four mice (2 males and 2 females) were averaged and are presented as means ± SEM. Tear secretion of WT and AQP4 KO animals did not differ significantly.

    Techniques Used:

    AQP4 immunofluorescence staining of LG from WT and AQP4 KO mice. ( A, B ) AQP4 staining was most prominent in the ducts. Robust AQP4 staining could be observed in the basolateral membrane of duct cells ( arrows ), and to a lesser extent in the apical side of the ducts and in the cytoplasmic area. ( C ) No AQP4 staining could be detected in LG tissues from AQP4 KO animals.
    Figure Legend Snippet: AQP4 immunofluorescence staining of LG from WT and AQP4 KO mice. ( A, B ) AQP4 staining was most prominent in the ducts. Robust AQP4 staining could be observed in the basolateral membrane of duct cells ( arrows ), and to a lesser extent in the apical side of the ducts and in the cytoplasmic area. ( C ) No AQP4 staining could be detected in LG tissues from AQP4 KO animals.

    Techniques Used: Immunofluorescence, Staining, Membrane

    VPAC1 (panels A and B ) and VPAC2 (panels C and D ) immunofluorescence staining of LG from WT and AQP4 KO mice. Panel ( A ) VPAC1 staining in WT LG, panel ( B ) VPAC1 in KO LG. Panel ( C ) VPAC2 in WT LG, panel ( D ) VPAC2 in KO LG. The staining pattern proved to be similar in both WT and KO mouse LGs.
    Figure Legend Snippet: VPAC1 (panels A and B ) and VPAC2 (panels C and D ) immunofluorescence staining of LG from WT and AQP4 KO mice. Panel ( A ) VPAC1 staining in WT LG, panel ( B ) VPAC1 in KO LG. Panel ( C ) VPAC2 in WT LG, panel ( D ) VPAC2 in KO LG. The staining pattern proved to be similar in both WT and KO mouse LGs.

    Techniques Used: Immunofluorescence, Staining

    Osmotic (filtration) permeability of isolated LG ducts from WT and AQP4 KO mice. Interlobular and intralobar ducts were isolated from the LGs of WT and AQP4 KO mice. Changes in the relative luminal volume (V r ) induced by a 50% reduction in the osmolarity of the perfusate are shown. Measurements were performed by video-microscopy, capturing images at 5 second intervals. Data were obtained from six ducts isolated from three different animals and are presented as means ± SEM. No statistically significant difference could be detected in the filtration permeability between WT or AQP4 KO ducts.
    Figure Legend Snippet: Osmotic (filtration) permeability of isolated LG ducts from WT and AQP4 KO mice. Interlobular and intralobar ducts were isolated from the LGs of WT and AQP4 KO mice. Changes in the relative luminal volume (V r ) induced by a 50% reduction in the osmolarity of the perfusate are shown. Measurements were performed by video-microscopy, capturing images at 5 second intervals. Data were obtained from six ducts isolated from three different animals and are presented as means ± SEM. No statistically significant difference could be detected in the filtration permeability between WT or AQP4 KO ducts.

    Techniques Used: Filtration, Permeability, Isolation, Microscopy

    Effect of carbachol and VIP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to either 100 µM carbachol or 200 nM VIP. Secretory rates are shown. In both cases (carbachol and VIP) data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Carbachol stimulation was not affected by the absences of AQP4 channels, whereas the effect of VIP stimulation was significantly lower in AQP4 KO mouse ducts.
    Figure Legend Snippet: Effect of carbachol and VIP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to either 100 µM carbachol or 200 nM VIP. Secretory rates are shown. In both cases (carbachol and VIP) data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Carbachol stimulation was not affected by the absences of AQP4 channels, whereas the effect of VIP stimulation was significantly lower in AQP4 KO mouse ducts.

    Techniques Used: Isolation

    Effect of 100 µM 8-bromo cAMP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to 100 µM 8-bromo cAMP. Secretory rates are shown. Data were obtained from seven ducts isolated from four different animals and are presented as means ± SEM. Fluid secretory capability was significantly reduced in AQP4 KO mouse ducts.
    Figure Legend Snippet: Effect of 100 µM 8-bromo cAMP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to 100 µM 8-bromo cAMP. Secretory rates are shown. Data were obtained from seven ducts isolated from four different animals and are presented as means ± SEM. Fluid secretory capability was significantly reduced in AQP4 KO mouse ducts.

    Techniques Used: Isolation

    Effect of 10 µM phenylephrine on fluid secretion in mouse ducts isolated from WT and AQP4 KO LGs. WT and AQP4 KO ducts were exposed to 10 µM phenylephrine. Secretory rates are shown. Data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Secretory rate was significantly lower in ducts isolated from AQP4 KO animals.
    Figure Legend Snippet: Effect of 10 µM phenylephrine on fluid secretion in mouse ducts isolated from WT and AQP4 KO LGs. WT and AQP4 KO ducts were exposed to 10 µM phenylephrine. Secretory rates are shown. Data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Secretory rate was significantly lower in ducts isolated from AQP4 KO animals.

    Techniques Used: Isolation

    rabbit polyclonal anti calhm1  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti calhm1
    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of <t>CALHM1</t> channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test
    Rabbit Polyclonal Anti Calhm1, 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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    Images

    1) Product Images from "Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels"

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    Journal: Biological Research

    doi: 10.1186/s40659-024-00503-3

    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test
    Figure Legend Snippet: The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

    Techniques Used: Activation Assay, Inhibition, Fluorescence, Blocking Assay

    The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA
    Figure Legend Snippet: The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

    Techniques Used: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

    Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test
    Figure Legend Snippet: Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

    Techniques Used: Proximity Ligation Assay, Negative Control, Western Blot, Inhibition, Immunoprecipitation

    NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)
    Figure Legend Snippet: NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

    Techniques Used: Expressing, Injection, Membrane, Western Blot

    Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)
    Figure Legend Snippet: Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

    Techniques Used: Activation Assay, Activity Assay, Membrane

    rabbit polyclonal primary antibody anti calhm1  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit polyclonal primary antibody anti calhm1
    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of <t>CALHM1</t> channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test
    Rabbit Polyclonal Primary Antibody Anti Calhm1, 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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    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels"

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    Journal: Biological Research

    doi: 10.1186/s40659-024-00503-3

    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test
    Figure Legend Snippet: The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

    Techniques Used: Activation Assay, Inhibition, Fluorescence, Blocking Assay

    The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA
    Figure Legend Snippet: The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

    Techniques Used: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

    Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test
    Figure Legend Snippet: Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

    Techniques Used: Proximity Ligation Assay, Negative Control, Western Blot, Inhibition, Immunoprecipitation

    NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)
    Figure Legend Snippet: NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

    Techniques Used: Expressing, Injection, Membrane, Western Blot

    Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)
    Figure Legend Snippet: Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

    Techniques Used: Activation Assay, Activity Assay, Membrane

    rabbit anti α3 gaba a r subunit extracellular polyclonal  (Alomone Labs)


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    Alomone Labs rabbit anti α3 gaba a r subunit extracellular polyclonal
    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of <t>α3-GABA</t> A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the <t>GABA</t> <t>A</t> <t>R</t> agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
    Rabbit Anti α3 Gaba A R Subunit Extracellular 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
    https://www.bioz.com/result/rabbit anti α3 gaba a r subunit extracellular polyclonal/product/Alomone Labs
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    Images

    1) Product Images from "GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors"

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.113834

    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
    Figure Legend Snippet: (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).

    Techniques Used: Confocal Microscopy, Labeling, Membrane, Immunolabeling, Expressing

    (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).
    Figure Legend Snippet: (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).

    Techniques Used: Inhibition


    Figure Legend Snippet:

    Techniques Used: Plasmid Preparation, Electron Microscopy, Blocking Assay, Virus, Recombinant, Saline, Microscopy, Software, Imaging

    rabbit anti gaba a r α 3 extracellular polyclonal  (Alomone Labs)


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    Alomone Labs rabbit anti gaba a r α 3 extracellular polyclonal
    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the <t>GABA</t> <t>A</t> <t>R</t> agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
    Rabbit Anti Gaba A R α 3 Extracellular 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
    https://www.bioz.com/result/rabbit anti gaba a r α 3 extracellular polyclonal/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti gaba a r α 3 extracellular polyclonal - by Bioz Stars, 2024-06
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    1) Product Images from "GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors"

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.113834

    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
    Figure Legend Snippet: (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).

    Techniques Used: Confocal Microscopy, Labeling, Membrane, Immunolabeling, Expressing

    (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).
    Figure Legend Snippet: (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).

    Techniques Used: Inhibition


    Figure Legend Snippet:

    Techniques Used: Plasmid Preparation, Electron Microscopy, Blocking Assay, Virus, Recombinant, Saline, Microscopy, Software, Imaging

    polyclonal rabbit anti aquaporin 4  (Alomone Labs)


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    Alomone Labs polyclonal rabbit anti aquaporin 4
    Polyclonal Rabbit Anti Aquaporin 4, 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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    rabbit polyclonal igg anti ca v 1 2  (Alomone Labs)


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    Alomone Labs rabbit polyclonal igg anti ca v 1 2
    a Single-molecule localization microscopy (SMLM) map showing Ca V 1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca V 1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c , d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in ( b , d ) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.
    Rabbit Polyclonal Igg Anti Ca V 1 2, 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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    1) Product Images from "BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts"

    Article Title: BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

    Journal: Nature Communications

    doi: 10.1038/s41467-024-47847-8

    a Single-molecule localization microscopy (SMLM) map showing Ca V 1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca V 1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c , d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in ( b , d ) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.
    Figure Legend Snippet: a Single-molecule localization microscopy (SMLM) map showing Ca V 1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca V 1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c , d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in ( b , d ) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Techniques Used: Microscopy, Comparison

    a Airyscan super-resolution images of Ca V 1.2 (green) and EEA1 (magenta) immunostained myocytes with and without ISO. Bottom: Binary colocalization maps show pixels in which Ca V 1.2 and EEA1 completely overlapped. b dot-plots summarizing % colocalization between EEA1 and Ca V 1.2 young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 14) myocytes, and c EEA1-positive endosome areas in young (control: N = 3, n = 15; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 13) myocytes. Data were analyzed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b , c ) are from JAX young. Note no significant differences in EEA1/Ca V 1.2 colocalization, responsivity to ISO, or endosome size was detected when young JAX and young NIA myocytes were compared (Supplementary Fig.  ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.
    Figure Legend Snippet: a Airyscan super-resolution images of Ca V 1.2 (green) and EEA1 (magenta) immunostained myocytes with and without ISO. Bottom: Binary colocalization maps show pixels in which Ca V 1.2 and EEA1 completely overlapped. b dot-plots summarizing % colocalization between EEA1 and Ca V 1.2 young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 14) myocytes, and c EEA1-positive endosome areas in young (control: N = 3, n = 15; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 13) myocytes. Data were analyzed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b , c ) are from JAX young. Note no significant differences in EEA1/Ca V 1.2 colocalization, responsivity to ISO, or endosome size was detected when young JAX and young NIA myocytes were compared (Supplementary Fig. ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Techniques Used: Comparison

    a Representative whole-cell currents from shRNA-scrmb and shRNA-mBIN1 myocytes before and during ISO application. b fold change in peak I Ca with ISO in young, old, shRNA-scrmb ( N = 3, n = 7), and shRNA-mBIN1 ( N = 3, n = 9) myocytes. c Representative Ca 2+ transients recorded from old shRNA-scrmb and shRNA-mBIN1 myocytes before and after ISO.  Fold increase after ISO from young, old, shRNA-scrmb ( N = 3, n = 14), and shRNA-mBIN1 ( N = 5, n = 18) myocytes. e SMLM localization maps showing Ca V 1.2 channel localization on t-tubules of myocytes from old shRNA-scrmb and shRNA-mBIN1, with or without ISO stimulation. Regions of interest are highlighted by yellow boxes. f Fold change in mean Ca V 1.2 channel cluster area with ISO in the young, old, old shRNA-scrmb (control: N = 3, n = 14; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 12; ISO: N = 3, n = 11). g , h show the same layout for RyR2 immunostained old shRNA-scrmb (control: N = 3, n = 12; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 9; ISO: N = 3, n = 8). Old and young data points in ( b ,  , f , h ) are reproduced from data in Figs.  b, h, and  b,  respectively. Statistical analysis was performed on data in ( b ,  , f , h ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b ) are pooled from NIA young and JAX young myocytes, data in (  , f , h ) are from NIA young myocytes. Note there was no significant difference in I Ca , Ca V 1.2, and RyR2 cluster areas when JAX and NIA young mice were compared (see Supplementary Fig.  ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.
    Figure Legend Snippet: a Representative whole-cell currents from shRNA-scrmb and shRNA-mBIN1 myocytes before and during ISO application. b fold change in peak I Ca with ISO in young, old, shRNA-scrmb ( N = 3, n = 7), and shRNA-mBIN1 ( N = 3, n = 9) myocytes. c Representative Ca 2+ transients recorded from old shRNA-scrmb and shRNA-mBIN1 myocytes before and after ISO. Fold increase after ISO from young, old, shRNA-scrmb ( N = 3, n = 14), and shRNA-mBIN1 ( N = 5, n = 18) myocytes. e SMLM localization maps showing Ca V 1.2 channel localization on t-tubules of myocytes from old shRNA-scrmb and shRNA-mBIN1, with or without ISO stimulation. Regions of interest are highlighted by yellow boxes. f Fold change in mean Ca V 1.2 channel cluster area with ISO in the young, old, old shRNA-scrmb (control: N = 3, n = 14; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 12; ISO: N = 3, n = 11). g , h show the same layout for RyR2 immunostained old shRNA-scrmb (control: N = 3, n = 12; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 9; ISO: N = 3, n = 8). Old and young data points in ( b , , f , h ) are reproduced from data in Figs. b, h, and b, respectively. Statistical analysis was performed on data in ( b , , f , h ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b ) are pooled from NIA young and JAX young myocytes, data in ( , f , h ) are from NIA young myocytes. Note there was no significant difference in I Ca , Ca V 1.2, and RyR2 cluster areas when JAX and NIA young mice were compared (see Supplementary Fig. ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Techniques Used: shRNA, Comparison

    The main findings of our study graphically illustrated and summarized. Top : In healthy young cells, Ca V 1.2 channels undergo endosomal recycling, where channels on endosomes are either marked for degradation through the late endosome pathway or are recycled to the sarcolemma through the fast and slow recycling pathways. Following β -adrenergic receptor ( β -AR) stimulation, a pool of channels localized to endosomes are mobilized to the membrane, resulting in larger Ca V 1.2 clusters along t-tubules. Across the dyad, RyR2 clusters on the sarcoplasmic reticulum also increase following βAR stimulation ensuring efficient Ca 2+ -induced Ca 2+ -release. This increase in cytosolic Ca 2+ , along with increased phosphorylation of cardiac Troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) within the sarcomere, allows for enhanced contractility under acute stress to cope with elevated hemodynamic and metabolic demands. Bottom : In aging, Bridging Integrator 1 (BIN1) protein levels are increased, accompanied by a swelling of endosomes and subsequent dysregulation of endosomal trafficking of Ca V 1.2. Ca V 1.2 and RyR2 channels are basally super-clustered at the dyads and lose β -AR responsivity. Reduced phosphorylation of cTnI and cMyBP-C result in systolic and diastolic dysfunction. BIN1 knockdown in aging recovers RyR2 clustering plasticity and Ca 2+ transient responsivity to β- AR stimulation. Phosphorylation of cMyBP-C is basally restored, and contractility is recovered to youthful levels. Thus, BIN1 knockdown rejuvenates the aging heart. Created with BioRender.com.
    Figure Legend Snippet: The main findings of our study graphically illustrated and summarized. Top : In healthy young cells, Ca V 1.2 channels undergo endosomal recycling, where channels on endosomes are either marked for degradation through the late endosome pathway or are recycled to the sarcolemma through the fast and slow recycling pathways. Following β -adrenergic receptor ( β -AR) stimulation, a pool of channels localized to endosomes are mobilized to the membrane, resulting in larger Ca V 1.2 clusters along t-tubules. Across the dyad, RyR2 clusters on the sarcoplasmic reticulum also increase following βAR stimulation ensuring efficient Ca 2+ -induced Ca 2+ -release. This increase in cytosolic Ca 2+ , along with increased phosphorylation of cardiac Troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) within the sarcomere, allows for enhanced contractility under acute stress to cope with elevated hemodynamic and metabolic demands. Bottom : In aging, Bridging Integrator 1 (BIN1) protein levels are increased, accompanied by a swelling of endosomes and subsequent dysregulation of endosomal trafficking of Ca V 1.2. Ca V 1.2 and RyR2 channels are basally super-clustered at the dyads and lose β -AR responsivity. Reduced phosphorylation of cTnI and cMyBP-C result in systolic and diastolic dysfunction. BIN1 knockdown in aging recovers RyR2 clustering plasticity and Ca 2+ transient responsivity to β- AR stimulation. Phosphorylation of cMyBP-C is basally restored, and contractility is recovered to youthful levels. Thus, BIN1 knockdown rejuvenates the aging heart. Created with BioRender.com.

    Techniques Used: Membrane, Binding Assay

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    Alomone Labs anti ca v 1 3 polyclonal rabbit antibody
    a Confocal images showing an overlay of Halo-Ca V 1.3 (JF646-HTL, magenta) with either PM (green) or F-actin signals (green) generated by three-color co-staining. b Orthogonal XZ image of a cell expressing Halo-Ca V 1.3 (magenta) and GFP- Rab 11a (green). Two principal imaging planes used for colocalization analysis are highlighted by white boxes: Medial cell sectioning (1) used for protein localization in ( a , c ), basal plane imaging (2) used for quantitative colocalization analysis in ( d ). c Confocal images of cells co-expressing Halo-Ca V 1.3 with GFP- Rab 4a, GFP- Rab 5a, and GFP- Rab 11a, respectively. d Representative segmentation maps of cell surface localized Halo-Ca V 1.3 signals (magenta, STED) together with each indicated endosomal marker (green, confocal). Colocalization is shown in black. Scale bars: 5 µm ( a – c ), 1 µm ( d ).
    Anti Ca V 1 3 Polyclonal Rabbit Antibody, 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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    Alomone Labs polyclonal rabbit anti trpc3 antibody
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    Alomone Labs primary polyclonal rabbit anti trpc6 antibody
    Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or <t>TRPC6</t> siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.
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    Alomone Labs aqp4 primary rabbit polyclonal antibody
    Tear secretion of WT and <t>AQP4</t> KO mice. Data from both eyes of four mice (2 males and 2 females) were averaged and are presented as means ± SEM. Tear secretion of WT and AQP4 KO animals did not differ significantly.
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    Alomone Labs rabbit polyclonal anti calhm1
    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of <t>CALHM1</t> channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test
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    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of <t>CALHM1</t> channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test
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    Alomone Labs rabbit anti α3 gaba a r subunit extracellular polyclonal
    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of <t>α3-GABA</t> A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the <t>GABA</t> <t>A</t> <t>R</t> agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
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    Alomone Labs rabbit anti gaba a r α 3 extracellular polyclonal
    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the <t>GABA</t> <t>A</t> <t>R</t> agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
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    Alomone Labs polyclonal rabbit anti aquaporin 4
    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the <t>GABA</t> <t>A</t> <t>R</t> agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).
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    Alomone Labs rabbit polyclonal igg anti ca v 1 2
    a Single-molecule localization microscopy (SMLM) map showing Ca V 1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca V 1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c , d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in ( b , d ) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.
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    Image Search Results


    a Confocal images showing an overlay of Halo-Ca V 1.3 (JF646-HTL, magenta) with either PM (green) or F-actin signals (green) generated by three-color co-staining. b Orthogonal XZ image of a cell expressing Halo-Ca V 1.3 (magenta) and GFP- Rab 11a (green). Two principal imaging planes used for colocalization analysis are highlighted by white boxes: Medial cell sectioning (1) used for protein localization in ( a , c ), basal plane imaging (2) used for quantitative colocalization analysis in ( d ). c Confocal images of cells co-expressing Halo-Ca V 1.3 with GFP- Rab 4a, GFP- Rab 5a, and GFP- Rab 11a, respectively. d Representative segmentation maps of cell surface localized Halo-Ca V 1.3 signals (magenta, STED) together with each indicated endosomal marker (green, confocal). Colocalization is shown in black. Scale bars: 5 µm ( a – c ), 1 µm ( d ).

    Journal: Communications Biology

    Article Title: Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

    doi: 10.1038/s42003-024-06313-3

    Figure Lengend Snippet: a Confocal images showing an overlay of Halo-Ca V 1.3 (JF646-HTL, magenta) with either PM (green) or F-actin signals (green) generated by three-color co-staining. b Orthogonal XZ image of a cell expressing Halo-Ca V 1.3 (magenta) and GFP- Rab 11a (green). Two principal imaging planes used for colocalization analysis are highlighted by white boxes: Medial cell sectioning (1) used for protein localization in ( a , c ), basal plane imaging (2) used for quantitative colocalization analysis in ( d ). c Confocal images of cells co-expressing Halo-Ca V 1.3 with GFP- Rab 4a, GFP- Rab 5a, and GFP- Rab 11a, respectively. d Representative segmentation maps of cell surface localized Halo-Ca V 1.3 signals (magenta, STED) together with each indicated endosomal marker (green, confocal). Colocalization is shown in black. Scale bars: 5 µm ( a – c ), 1 µm ( d ).

    Article Snippet: The following antibodies were used: Anti-Ca V 1.3 polyclonal rabbit antibody (Alomone Labs ACC-005, dilution 1:100) and anti-Ca V 1.3 mouse monoclonal antibody (Aviva Biosystems OASE00151, dilution 1:200) both raised against AA 859-875 of rat Ca V 1.3.

    Techniques: Generated, Staining, Expressing, Imaging, Marker

    a Live-cell confocal images of transiently transfected HEK293 cells show cell-surface localized signals of Halo-Ca V 1.3 channels labeled with JF646-HTL (‘Fire’ LUT). The lower-hand image demonstrates high signal densities in the basal PM imaging plane. b Cell-surface STED nanoscopy revealed a clustered distribution of Halo-Ca V 1.3 signals, as resolved by STED but not by confocal imaging. Individual cluster signals were segmented as shown by white outlines. For each cluster, the signal brightness was corrected for the local background prior to molecular counting by brightness referencing. c Frequency distribution of the cluster area obtained from segmented signals ( n clusters = 9459 clusters from n cell = 75). The values in blue provide the corresponding diameter of the clusters assuming a round cluster shape. The figure legend shows the mean and s.d. for the cluster area and diameter. d Frequency distribution of fluorescent channel counts within the segmented cluster signals as determined by brightness referencing. e Scatter graph showing the relationship between fluorescent channel counts and cluster area. The correlation was quantified by Spearman’s r = 0.87 ( p < 0.0001). f Scatter graph of the molecular density and cluster area with r = 0.18 ( p < 0.0001). Scale bars: 20 µm ( a ), 1 µm ( b ).

    Journal: Communications Biology

    Article Title: Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

    doi: 10.1038/s42003-024-06313-3

    Figure Lengend Snippet: a Live-cell confocal images of transiently transfected HEK293 cells show cell-surface localized signals of Halo-Ca V 1.3 channels labeled with JF646-HTL (‘Fire’ LUT). The lower-hand image demonstrates high signal densities in the basal PM imaging plane. b Cell-surface STED nanoscopy revealed a clustered distribution of Halo-Ca V 1.3 signals, as resolved by STED but not by confocal imaging. Individual cluster signals were segmented as shown by white outlines. For each cluster, the signal brightness was corrected for the local background prior to molecular counting by brightness referencing. c Frequency distribution of the cluster area obtained from segmented signals ( n clusters = 9459 clusters from n cell = 75). The values in blue provide the corresponding diameter of the clusters assuming a round cluster shape. The figure legend shows the mean and s.d. for the cluster area and diameter. d Frequency distribution of fluorescent channel counts within the segmented cluster signals as determined by brightness referencing. e Scatter graph showing the relationship between fluorescent channel counts and cluster area. The correlation was quantified by Spearman’s r = 0.87 ( p < 0.0001). f Scatter graph of the molecular density and cluster area with r = 0.18 ( p < 0.0001). Scale bars: 20 µm ( a ), 1 µm ( b ).

    Article Snippet: The following antibodies were used: Anti-Ca V 1.3 polyclonal rabbit antibody (Alomone Labs ACC-005, dilution 1:100) and anti-Ca V 1.3 mouse monoclonal antibody (Aviva Biosystems OASE00151, dilution 1:200) both raised against AA 859-875 of rat Ca V 1.3.

    Techniques: Transfection, Labeling, Imaging

    a Schematics of the production of GPMVs derived from cells and the spreading of GPMVs on a solid support. b Confocal image of the Halo-Ca V 1.3 signal (‘Fire’ LUT) showing a GPMV attached to a HEK293 cell. c Upright STED image resolving spot-like fluorescent Halo-Ca V 1.3 clusters in the detached GPMV. d Upright STED image of a supported plasma membrane bilayer (SPMB) obtained by spreading a GPMV on soda lime-glass (left image: R18, red; right image: Halo-Ca V 1.3, magenta). e Distribution of the SPMB surface areas with a mean area of 410 ± 340 µm² (mean ± s.d.) ( n SPMB = 232). Scale bars: 5 µm ( b ), 10 µm ( c , d ).

    Journal: Communications Biology

    Article Title: Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

    doi: 10.1038/s42003-024-06313-3

    Figure Lengend Snippet: a Schematics of the production of GPMVs derived from cells and the spreading of GPMVs on a solid support. b Confocal image of the Halo-Ca V 1.3 signal (‘Fire’ LUT) showing a GPMV attached to a HEK293 cell. c Upright STED image resolving spot-like fluorescent Halo-Ca V 1.3 clusters in the detached GPMV. d Upright STED image of a supported plasma membrane bilayer (SPMB) obtained by spreading a GPMV on soda lime-glass (left image: R18, red; right image: Halo-Ca V 1.3, magenta). e Distribution of the SPMB surface areas with a mean area of 410 ± 340 µm² (mean ± s.d.) ( n SPMB = 232). Scale bars: 5 µm ( b ), 10 µm ( c , d ).

    Article Snippet: The following antibodies were used: Anti-Ca V 1.3 polyclonal rabbit antibody (Alomone Labs ACC-005, dilution 1:100) and anti-Ca V 1.3 mouse monoclonal antibody (Aviva Biosystems OASE00151, dilution 1:200) both raised against AA 859-875 of rat Ca V 1.3.

    Techniques: Derivative Assay, Membrane

    a SPMB (R18, red) obtained upon spreading a GPMV on borosilicate glass (left image, confocal). The SPMB contains Halo-Ca V 1.3 channel clusters (‘Fire’ LUT) (right image). The bottom part shows the confocal image, and the top part is the STED image. b Comparison of the appearance of the Halo-Ca V 1.3 clusters in SPMBs and in living cells. Single Halo-Ca V 1.3 clusters are resolved, allowing us to determine the c cluster area, d the channel counts, and e the molecular density (box: IQR, dot: mean, line: med.; whiskers: 5–95%) [Mann–Whitney U -test: **** P ≤ 0.0001]. n is the number of analyzed clusters. f , g Two-dimensional kernel density (2D-KDF) of Ca V 1.3 cluster properties of cells (blue) and SPMBs (black). f 2D-KDF of the channel counts vs. cluster area and g 2D-KDF of the molecular density vs. cluster area ( n cell = 75, n SPMB = 20). Scale bars: 10 µm ( a ); 500 nm ( b ).

    Journal: Communications Biology

    Article Title: Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

    doi: 10.1038/s42003-024-06313-3

    Figure Lengend Snippet: a SPMB (R18, red) obtained upon spreading a GPMV on borosilicate glass (left image, confocal). The SPMB contains Halo-Ca V 1.3 channel clusters (‘Fire’ LUT) (right image). The bottom part shows the confocal image, and the top part is the STED image. b Comparison of the appearance of the Halo-Ca V 1.3 clusters in SPMBs and in living cells. Single Halo-Ca V 1.3 clusters are resolved, allowing us to determine the c cluster area, d the channel counts, and e the molecular density (box: IQR, dot: mean, line: med.; whiskers: 5–95%) [Mann–Whitney U -test: **** P ≤ 0.0001]. n is the number of analyzed clusters. f , g Two-dimensional kernel density (2D-KDF) of Ca V 1.3 cluster properties of cells (blue) and SPMBs (black). f 2D-KDF of the channel counts vs. cluster area and g 2D-KDF of the molecular density vs. cluster area ( n cell = 75, n SPMB = 20). Scale bars: 10 µm ( a ); 500 nm ( b ).

    Article Snippet: The following antibodies were used: Anti-Ca V 1.3 polyclonal rabbit antibody (Alomone Labs ACC-005, dilution 1:100) and anti-Ca V 1.3 mouse monoclonal antibody (Aviva Biosystems OASE00151, dilution 1:200) both raised against AA 859-875 of rat Ca V 1.3.

    Techniques: Comparison, MANN-WHITNEY

    a SPMB with Halo-Ca V 1.3 clusters (labeled with JF646-HTL, ‘Fire’ LUT, brightness scale identical for both images) without (no ISO) and with (ISO) treatment. b Cluster density as a function of the channel counts (mean ± s.e.m.), (untreated SPMBs, black; ISO-treated SPMBs, red). c Two-dimensional kernel densities (2D-KDF) of the channel counts as a function of cluster area for untreated SPMBs (black) and ISO-treated SPMBs (red). d 2D-KDF of the molecular density vs. cluster area for untreated (no ISO, black) and ISO-treated (ISO, red) SPMBs. Reproduced in four experiments with n SPMB,no ISO = 20, n clusters,no ISO = 9862; n SPMB,ISO = 17, n clusters,ISO = 5818. Scale bar: 1 µm ( a ).

    Journal: Communications Biology

    Article Title: Ca V 1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

    doi: 10.1038/s42003-024-06313-3

    Figure Lengend Snippet: a SPMB with Halo-Ca V 1.3 clusters (labeled with JF646-HTL, ‘Fire’ LUT, brightness scale identical for both images) without (no ISO) and with (ISO) treatment. b Cluster density as a function of the channel counts (mean ± s.e.m.), (untreated SPMBs, black; ISO-treated SPMBs, red). c Two-dimensional kernel densities (2D-KDF) of the channel counts as a function of cluster area for untreated SPMBs (black) and ISO-treated SPMBs (red). d 2D-KDF of the molecular density vs. cluster area for untreated (no ISO, black) and ISO-treated (ISO, red) SPMBs. Reproduced in four experiments with n SPMB,no ISO = 20, n clusters,no ISO = 9862; n SPMB,ISO = 17, n clusters,ISO = 5818. Scale bar: 1 µm ( a ).

    Article Snippet: The following antibodies were used: Anti-Ca V 1.3 polyclonal rabbit antibody (Alomone Labs ACC-005, dilution 1:100) and anti-Ca V 1.3 mouse monoclonal antibody (Aviva Biosystems OASE00151, dilution 1:200) both raised against AA 859-875 of rat Ca V 1.3.

    Techniques: Labeling

    L687, a TRPC3/C6/C7 activator, induces intracellular Ca 2+ uptake. ( A ) Analysis of intracellular Ca 2+ influx with various concentrations of L687 or PPZ2 in HEK293 cells, overexpressing TRPC3/C6/C7, respectively, in the presence of 2 mM Ca 2+ . Ca 2+ influx was recorded following the addition of each compound. ( B ) Analysis of Ca 2+ influx into the A549 cells in the presence of 2 mM Ca 2+ . After adding PPZ2 (30 μM) or L687 (3, 10 and 30 μM), the Ca 2+ influx was analysed. TRPC, transient receptor potential canonical.

    Journal: Nucleic Acids Research

    Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

    doi: 10.1093/nar/gkae245

    Figure Lengend Snippet: L687, a TRPC3/C6/C7 activator, induces intracellular Ca 2+ uptake. ( A ) Analysis of intracellular Ca 2+ influx with various concentrations of L687 or PPZ2 in HEK293 cells, overexpressing TRPC3/C6/C7, respectively, in the presence of 2 mM Ca 2+ . Ca 2+ influx was recorded following the addition of each compound. ( B ) Analysis of Ca 2+ influx into the A549 cells in the presence of 2 mM Ca 2+ . After adding PPZ2 (30 μM) or L687 (3, 10 and 30 μM), the Ca 2+ influx was analysed. TRPC, transient receptor potential canonical.

    Article Snippet: Incubation with primary polyclonal rabbit anti-TRPC3 antibody (#ACC-016; Alomone Labs, Jerusalem, Israel), primary polyclonal rabbit anti-TRPC6 antibody (#ACC-120; Alomone Labs, Jerusalem, Israel) at 1:2000 dilution and mouse monoclonal anti-GAPDH (#AM4300; Thermo Fisher Scientific, MA, USA) at 1:2000 dilution was performed at 4°C overnight.

    Techniques:

    Effects of the TRPC3/C6/C7 activator L687 or CBD on ASO activity. ( A ) Analysis of MALAT1 expression by adding MALAT1_ASO with L687. MALAT1_ASO (100 nM) was added to either 1–30 μM L687 in the medium. A549 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of REST versus GAPDH was compared with that of MALAT1 in untreated A549 cells. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of mock DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) Analysis of SRRM4 expression by adding SRRM4_ASO with L687. SRRM4_ASO (100 nM) was added with 30 μM L687 to the medium. N417 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of SRRM4 versus GAPDH was compared with that of SRRM4 in untreated N417 cells. ( C , D ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (100 nM) was added to 10 and 30 μM of L687 or CBD in the medium. A549 cells were collected after 48 h, and the relative expression of REST versus GAPDH was analysed. ( E ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (20 and 100 nM) was added to 30 μM L687 in the medium. A549 cells were collected after 72 h, and the relative expression of REST versus GAPDH was analysed. ( F ) Analysis of ERBB2 expression by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. SK-OV-3 cells were collected after 48 h, and the relative expression of ERBB2 versus β-actin was analysed. ( G ) Viability analysis of SK-OV-3 by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. Cell viability was analysed after 48 h of incubation. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative PCR; TRPC, transient receptor potential canonical.

    Journal: Nucleic Acids Research

    Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

    doi: 10.1093/nar/gkae245

    Figure Lengend Snippet: Effects of the TRPC3/C6/C7 activator L687 or CBD on ASO activity. ( A ) Analysis of MALAT1 expression by adding MALAT1_ASO with L687. MALAT1_ASO (100 nM) was added to either 1–30 μM L687 in the medium. A549 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of REST versus GAPDH was compared with that of MALAT1 in untreated A549 cells. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of mock DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) Analysis of SRRM4 expression by adding SRRM4_ASO with L687. SRRM4_ASO (100 nM) was added with 30 μM L687 to the medium. N417 cells were collected after 48 h, and total RNA was prepared for RT-qPCR analysis. The relative expression of SRRM4 versus GAPDH was compared with that of SRRM4 in untreated N417 cells. ( C , D ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (100 nM) was added to 10 and 30 μM of L687 or CBD in the medium. A549 cells were collected after 48 h, and the relative expression of REST versus GAPDH was analysed. ( E ) Analysis of REST expression by adding REST_ASO with L687. REST_ASO (20 and 100 nM) was added to 30 μM L687 in the medium. A549 cells were collected after 72 h, and the relative expression of REST versus GAPDH was analysed. ( F ) Analysis of ERBB2 expression by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. SK-OV-3 cells were collected after 48 h, and the relative expression of ERBB2 versus β-actin was analysed. ( G ) Viability analysis of SK-OV-3 by adding ERBB2_ASO with L687. ERBB2_ASO (100 nM) was added to 3–30 μM of L687 in the medium. Cell viability was analysed after 48 h of incubation. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative PCR; TRPC, transient receptor potential canonical.

    Article Snippet: Incubation with primary polyclonal rabbit anti-TRPC3 antibody (#ACC-016; Alomone Labs, Jerusalem, Israel), primary polyclonal rabbit anti-TRPC6 antibody (#ACC-120; Alomone Labs, Jerusalem, Israel) at 1:2000 dilution and mouse monoclonal anti-GAPDH (#AM4300; Thermo Fisher Scientific, MA, USA) at 1:2000 dilution was performed at 4°C overnight.

    Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction

    Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

    Journal: Nucleic Acids Research

    Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

    doi: 10.1093/nar/gkae245

    Figure Lengend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

    Article Snippet: Incubation with primary polyclonal rabbit anti-TRPC3 antibody (#ACC-016; Alomone Labs, Jerusalem, Israel), primary polyclonal rabbit anti-TRPC6 antibody (#ACC-120; Alomone Labs, Jerusalem, Israel) at 1:2000 dilution and mouse monoclonal anti-GAPDH (#AM4300; Thermo Fisher Scientific, MA, USA) at 1:2000 dilution was performed at 4°C overnight.

    Techniques: Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy

    Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

    Journal: Nucleic Acids Research

    Article Title: A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides

    doi: 10.1093/nar/gkae245

    Figure Lengend Snippet: Effects of the TRPC3/C6/C7 inhibitor, knockdown of TRPC3/C6, and Ca 2+ chelator on ASO uptake, and examination of L687-mediated uptake pathways. ( A ) ASO uptake was analysed by incubating cells with or without a TRPC inhibitor (SKF96365). Alexa647-AmNA#26 (10 nM) was added to either 10 μM L687, 20 μM GSK1702934A, or 30 μM CBD with or without 20 μM SKF96365 in the medium. After 24 h, the intracellular fluorescence intensities were analysed by flow cytometry. Data are shown as the relative MFI of ASO in DMSO. All data are presented as mean ± standard error of the mean (SEM) of three independent experiments ( n = 3). Statistical significance was determined by comparing with values of DMSO using Tukey's test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( B ) siRNA-mediated knockdown of the TRPC3/C6 channels. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed and compared with that in the untreated cells. ( C ) siRNA-mediated knockdown of TRPC3/C6 channel. TRPC3 siRNA (30 nM) or TRPC6 siRNA (10 nM) was transfected into A549 cells using Lipofectamine3000, and cells were collected after 48 h. The relative expression of TRPC3/C6 was analysed by western blot and compared with that in untreated cells. ( D ) The effects of siRNA-mediated TRPC3/C6 channel knockdown on ASO uptake. TRPC3 siRNA (30 nM), TRPC6 (10 nM), or a combination of both were transfected into A549 cells for 48 h. The medium was replaced with Alexa647-AmNA#26 containing L687, and intracellular fluorescence intensities were analysed after 24 h. Data are shown as the relative MFI of ASO in DMSO. ( E ) Analysis of ASO uptake after incubating cells with a Ca 2+ chelator (BAPTA-AM). ASO and L687 were added to the medium, with or without 10 μM BAPTA-AM. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( F ) Analysis of dextran uptake mediated by L687. Alexa647-labelled dextran (1 and 3 μM) with 10 and 30 μM L687 was added to the medium, and A549 cells were cultured for 24 h. Intracellular fluorescence was analysed by flow cytometry, and the relative MFI was compared with 1 μM of Alexa647-dextran with DMSO. ( G ) Analysis of ASO uptake by incubating the cells with a macropinocytosis inhibitor (Cytochalasin D). L687 (30 μM) was then added to the medium. The following day, cells were then washed twice with PBS and incubated with cytochalasin D in the medium for 1 h. Then, cells were washed twice with PBS and incubated with Alexa647-AmNA#26 (10 nM) and L687 (30 μM). After 4 h, intracellular fluorescence intensities were analysed by flow cytometry. ( H ) Analysis of ASO uptake by incubating cells with a macropinocytosis inhibitor (EIPA). ASO and L687 were added to the medium with or without 100 μM EIPA. After 24 h, intracellular fluorescence intensities were analysed by flow cytometry. ( I ) Fluorescence imaging analysis of ASO incorporated into cells. Alexa647-AmNA#26 (100 nM) and L687 (30 μM) were added to the medium, and staining with Lysotracker-green and Hoechst, fluorescence microscopy imaging, and image analysis were performed after 48 h. ASO, antisense oligonucleotide; CBD, cannabidiol; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity; TRPC, transient receptor potential canonical.

    Article Snippet: Incubation with primary polyclonal rabbit anti-TRPC3 antibody (#ACC-016; Alomone Labs, Jerusalem, Israel), primary polyclonal rabbit anti-TRPC6 antibody (#ACC-120; Alomone Labs, Jerusalem, Israel) at 1:2000 dilution and mouse monoclonal anti-GAPDH (#AM4300; Thermo Fisher Scientific, MA, USA) at 1:2000 dilution was performed at 4°C overnight.

    Techniques: Fluorescence, Flow Cytometry, Transfection, Expressing, Western Blot, Cell Culture, Incubation, Imaging, Staining, Microscopy

    Tear secretion of WT and AQP4 KO mice. Data from both eyes of four mice (2 males and 2 females) were averaged and are presented as means ± SEM. Tear secretion of WT and AQP4 KO animals did not differ significantly.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: Tear secretion of WT and AQP4 KO mice. Data from both eyes of four mice (2 males and 2 females) were averaged and are presented as means ± SEM. Tear secretion of WT and AQP4 KO animals did not differ significantly.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques:

    AQP4 immunofluorescence staining of LG from WT and AQP4 KO mice. ( A, B ) AQP4 staining was most prominent in the ducts. Robust AQP4 staining could be observed in the basolateral membrane of duct cells ( arrows ), and to a lesser extent in the apical side of the ducts and in the cytoplasmic area. ( C ) No AQP4 staining could be detected in LG tissues from AQP4 KO animals.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: AQP4 immunofluorescence staining of LG from WT and AQP4 KO mice. ( A, B ) AQP4 staining was most prominent in the ducts. Robust AQP4 staining could be observed in the basolateral membrane of duct cells ( arrows ), and to a lesser extent in the apical side of the ducts and in the cytoplasmic area. ( C ) No AQP4 staining could be detected in LG tissues from AQP4 KO animals.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques: Immunofluorescence, Staining, Membrane

    VPAC1 (panels A and B ) and VPAC2 (panels C and D ) immunofluorescence staining of LG from WT and AQP4 KO mice. Panel ( A ) VPAC1 staining in WT LG, panel ( B ) VPAC1 in KO LG. Panel ( C ) VPAC2 in WT LG, panel ( D ) VPAC2 in KO LG. The staining pattern proved to be similar in both WT and KO mouse LGs.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: VPAC1 (panels A and B ) and VPAC2 (panels C and D ) immunofluorescence staining of LG from WT and AQP4 KO mice. Panel ( A ) VPAC1 staining in WT LG, panel ( B ) VPAC1 in KO LG. Panel ( C ) VPAC2 in WT LG, panel ( D ) VPAC2 in KO LG. The staining pattern proved to be similar in both WT and KO mouse LGs.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques: Immunofluorescence, Staining

    Osmotic (filtration) permeability of isolated LG ducts from WT and AQP4 KO mice. Interlobular and intralobar ducts were isolated from the LGs of WT and AQP4 KO mice. Changes in the relative luminal volume (V r ) induced by a 50% reduction in the osmolarity of the perfusate are shown. Measurements were performed by video-microscopy, capturing images at 5 second intervals. Data were obtained from six ducts isolated from three different animals and are presented as means ± SEM. No statistically significant difference could be detected in the filtration permeability between WT or AQP4 KO ducts.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: Osmotic (filtration) permeability of isolated LG ducts from WT and AQP4 KO mice. Interlobular and intralobar ducts were isolated from the LGs of WT and AQP4 KO mice. Changes in the relative luminal volume (V r ) induced by a 50% reduction in the osmolarity of the perfusate are shown. Measurements were performed by video-microscopy, capturing images at 5 second intervals. Data were obtained from six ducts isolated from three different animals and are presented as means ± SEM. No statistically significant difference could be detected in the filtration permeability between WT or AQP4 KO ducts.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques: Filtration, Permeability, Isolation, Microscopy

    Effect of carbachol and VIP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to either 100 µM carbachol or 200 nM VIP. Secretory rates are shown. In both cases (carbachol and VIP) data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Carbachol stimulation was not affected by the absences of AQP4 channels, whereas the effect of VIP stimulation was significantly lower in AQP4 KO mouse ducts.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: Effect of carbachol and VIP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to either 100 µM carbachol or 200 nM VIP. Secretory rates are shown. In both cases (carbachol and VIP) data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Carbachol stimulation was not affected by the absences of AQP4 channels, whereas the effect of VIP stimulation was significantly lower in AQP4 KO mouse ducts.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques: Isolation

    Effect of 100 µM 8-bromo cAMP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to 100 µM 8-bromo cAMP. Secretory rates are shown. Data were obtained from seven ducts isolated from four different animals and are presented as means ± SEM. Fluid secretory capability was significantly reduced in AQP4 KO mouse ducts.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: Effect of 100 µM 8-bromo cAMP on fluid secretion in mouse ducts isolated from WT and AQP4 KO mouse LGs. WT and AQP4 KO mouse ducts were exposed to 100 µM 8-bromo cAMP. Secretory rates are shown. Data were obtained from seven ducts isolated from four different animals and are presented as means ± SEM. Fluid secretory capability was significantly reduced in AQP4 KO mouse ducts.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques: Isolation

    Effect of 10 µM phenylephrine on fluid secretion in mouse ducts isolated from WT and AQP4 KO LGs. WT and AQP4 KO ducts were exposed to 10 µM phenylephrine. Secretory rates are shown. Data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Secretory rate was significantly lower in ducts isolated from AQP4 KO animals.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: The Role of Aquaporin 4 in Lacrimal Gland Ductal Fluid Secretion in Mice

    doi: 10.1167/iovs.65.5.30

    Figure Lengend Snippet: Effect of 10 µM phenylephrine on fluid secretion in mouse ducts isolated from WT and AQP4 KO LGs. WT and AQP4 KO ducts were exposed to 10 µM phenylephrine. Secretory rates are shown. Data were obtained from six ducts isolated from four different animals and are presented as means ± SEM. Secretory rate was significantly lower in ducts isolated from AQP4 KO animals.

    Article Snippet: For immunostaining experiments, VPAC1 and VPAC2 primary rabbit polyclonal antibodies and Alexa Fluor 488 secondary goat anti-rabbit antibody was obtained from Abcam (Cambridge, UK), AQP4 primary rabbit polyclonal antibody derived from Alomone Labs (Jerusalem, Israel).

    Techniques: Isolation

    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

    Article Snippet: The following primary antibodies were used in Western blot, Immunofluorescence and PLA analysis: mouse monoclonal anti-Cx43 (BD Transduction Laboratories™, USA, Cat. #610,062), rabbit polyclonal anti-Panx-1 (Sigma-Aldrich, USA, Cat. #AV42783-50UG), rabbit polyclonal anti-CALHM1 (Alomone Labs, Israel, Cat. #ACC-101), rabbit polyclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,298), mouse monoclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,296), rabbit monoclonal anti-nNOS (Cell Signaling Technology, Cat. #4231), rabbit polyclonal anti-iNOS (Millipore, Germany, Cat. #ab5383), rabbit polyclonal anti-GFAP (Sigma Aldrich, USA, Cat. #G9269), mouse monoclonal anti-GFAP (Sigma Aldrich, USA, #G3893), rabbit polyclonal anti-β-actin (Sigma Aldrich, USA, Cat. #A3853) and rabbit polyclonal anti-SNO-Cys (Sigma Aldrich, USA, Cat. #N5411).

    Techniques: Activation Assay, Inhibition, Fluorescence, Blocking Assay

    The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

    Article Snippet: The following primary antibodies were used in Western blot, Immunofluorescence and PLA analysis: mouse monoclonal anti-Cx43 (BD Transduction Laboratories™, USA, Cat. #610,062), rabbit polyclonal anti-Panx-1 (Sigma-Aldrich, USA, Cat. #AV42783-50UG), rabbit polyclonal anti-CALHM1 (Alomone Labs, Israel, Cat. #ACC-101), rabbit polyclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,298), mouse monoclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,296), rabbit monoclonal anti-nNOS (Cell Signaling Technology, Cat. #4231), rabbit polyclonal anti-iNOS (Millipore, Germany, Cat. #ab5383), rabbit polyclonal anti-GFAP (Sigma Aldrich, USA, Cat. #G9269), mouse monoclonal anti-GFAP (Sigma Aldrich, USA, #G3893), rabbit polyclonal anti-β-actin (Sigma Aldrich, USA, Cat. #A3853) and rabbit polyclonal anti-SNO-Cys (Sigma Aldrich, USA, Cat. #N5411).

    Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

    Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

    Article Snippet: The following primary antibodies were used in Western blot, Immunofluorescence and PLA analysis: mouse monoclonal anti-Cx43 (BD Transduction Laboratories™, USA, Cat. #610,062), rabbit polyclonal anti-Panx-1 (Sigma-Aldrich, USA, Cat. #AV42783-50UG), rabbit polyclonal anti-CALHM1 (Alomone Labs, Israel, Cat. #ACC-101), rabbit polyclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,298), mouse monoclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,296), rabbit monoclonal anti-nNOS (Cell Signaling Technology, Cat. #4231), rabbit polyclonal anti-iNOS (Millipore, Germany, Cat. #ab5383), rabbit polyclonal anti-GFAP (Sigma Aldrich, USA, Cat. #G9269), mouse monoclonal anti-GFAP (Sigma Aldrich, USA, #G3893), rabbit polyclonal anti-β-actin (Sigma Aldrich, USA, Cat. #A3853) and rabbit polyclonal anti-SNO-Cys (Sigma Aldrich, USA, Cat. #N5411).

    Techniques: Proximity Ligation Assay, Negative Control, Western Blot, Inhibition, Immunoprecipitation

    NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

    Article Snippet: The following primary antibodies were used in Western blot, Immunofluorescence and PLA analysis: mouse monoclonal anti-Cx43 (BD Transduction Laboratories™, USA, Cat. #610,062), rabbit polyclonal anti-Panx-1 (Sigma-Aldrich, USA, Cat. #AV42783-50UG), rabbit polyclonal anti-CALHM1 (Alomone Labs, Israel, Cat. #ACC-101), rabbit polyclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,298), mouse monoclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,296), rabbit monoclonal anti-nNOS (Cell Signaling Technology, Cat. #4231), rabbit polyclonal anti-iNOS (Millipore, Germany, Cat. #ab5383), rabbit polyclonal anti-GFAP (Sigma Aldrich, USA, Cat. #G9269), mouse monoclonal anti-GFAP (Sigma Aldrich, USA, #G3893), rabbit polyclonal anti-β-actin (Sigma Aldrich, USA, Cat. #A3853) and rabbit polyclonal anti-SNO-Cys (Sigma Aldrich, USA, Cat. #N5411).

    Techniques: Expressing, Injection, Membrane, Western Blot

    Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

    Article Snippet: The following primary antibodies were used in Western blot, Immunofluorescence and PLA analysis: mouse monoclonal anti-Cx43 (BD Transduction Laboratories™, USA, Cat. #610,062), rabbit polyclonal anti-Panx-1 (Sigma-Aldrich, USA, Cat. #AV42783-50UG), rabbit polyclonal anti-CALHM1 (Alomone Labs, Israel, Cat. #ACC-101), rabbit polyclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,298), mouse monoclonal anti-eNOS (BD Transduction Laboratories™, USA, Cat. #610,296), rabbit monoclonal anti-nNOS (Cell Signaling Technology, Cat. #4231), rabbit polyclonal anti-iNOS (Millipore, Germany, Cat. #ab5383), rabbit polyclonal anti-GFAP (Sigma Aldrich, USA, Cat. #G9269), mouse monoclonal anti-GFAP (Sigma Aldrich, USA, #G3893), rabbit polyclonal anti-β-actin (Sigma Aldrich, USA, Cat. #A3853) and rabbit polyclonal anti-SNO-Cys (Sigma Aldrich, USA, Cat. #N5411).

    Techniques: Activation Assay, Activity Assay, Membrane

    The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: The Cx hemichannel- and Panx-1 channel-mediated Ca 2+ signaling depends on the activation of purinergic receptors. A Time course of the increase in [Ca 2+ ] i induced in primary cultures of astrocytes by 10 µM glutamate in control conditions and after the blockade of purinergic receptors with 100 µM PPADS or the inhibition of CALHM1 channels with 20 µM ruthenium red (RuR). B Analysis of the increase in ethidium uptake rate induced by glutamate in control conditions and in the presence of PPADS or RuR. The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity (expressed as arbitrary units, AU) along the time in basal conditions and during the stimulation with glutamate. C ATP release evoked by glutamate in control conditions and after the treatment with 100 µM N ω -nitro-L-arginine (L-NA) to inhibit NO production, the mimetic peptide 37,43 Gap27 (Gap27, 100 µM) to block hemichannels formed by Cx37 or Cx43, 10 Panx (100 µM) to block Panx1 channels or RuR. ATP was measured after 3 min of stimulation with glutamate. Numbers inside the bars indicate the n value. D Time course of the increase in [Ca 2+ ] i elicited by 100 nM ATP in primary cultures of astrocytes in control conditions and in the presence of 37,43 Gap27 or 10 Panx. Values are means ± SEM. *P < 0.05 vs Control by one-way ANOVA plus Bonferroni post hoc test. † P < 0.05 vs Baseline by paired Student’s t-test

    Article Snippet: The sections were blocked with 0.5% BSA in PBS, incubated with a rabbit polyclonal primary antibody anti-CALHM1 (Alomone Labs, Israel) and a mouse monoclonal anti-GFAP (Sigma Aldrich, USA), and then, with an Alexa-568-labeled goat anti-rabbit secondary antibody and an Alexa-488-labeled goat anti-mouse secondary antibody (Invitrogen Molecular Probes, USA) using the Signal Enhancer HIKARI (Nacalai Tesque, INC, Japan) as indicated by the manufacturer.

    Techniques: Activation Assay, Inhibition, Fluorescence, Blocking Assay

    The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: The increase in [Ca 2+ ] i , activation of Cx hemichannels and Panx-1 channels and ATP release induced by glutamate depends on the expression of CALHM1 channels. A Detection of the expression of CALMH1 (red) by co-immunofluorescence analysis with glial fibrillary acidic protein (GFAP, green) in primary cultures of astrocytes treated with a control siRNA (siControl) or a siRNA directed against Calhm1 (siCALHM1). The cell nuclei are highlighted by the staining with DAPI (blue). B – D Representative Western blot and densitometric analysis of CALHM1 ( B ), Cx43 ( C ) and Panx-1 ( D ) expression in the primary cultures of astrocytes shown in A . Numbers inside the bars indicate the n value. The whole images of the representative Western blots are shown in Additional file : Fig. S7. E , Maximal increase in [Ca 2+ ] i observed in response to 10 µM glutamate in astrocytes cultures treated with siControl or siCALHM1. F Time course of the increase in ethidium uptake induced by 10 µM glutamate in primary cultures of astrocytes treated with siControl or siCALHM1. The horizontal bar indicates the stimulation period. G ATP release attained 3 min after the stimulation with glutamate in the same conditions than those shown in E and F . Values are means ± SEM. *P < 0.05 vs siControl by unpaired Student’s t-test. † P < 0.05 vs siControl by two-way ANOVA

    Article Snippet: The sections were blocked with 0.5% BSA in PBS, incubated with a rabbit polyclonal primary antibody anti-CALHM1 (Alomone Labs, Israel) and a mouse monoclonal anti-GFAP (Sigma Aldrich, USA), and then, with an Alexa-568-labeled goat anti-rabbit secondary antibody and an Alexa-488-labeled goat anti-mouse secondary antibody (Invitrogen Molecular Probes, USA) using the Signal Enhancer HIKARI (Nacalai Tesque, INC, Japan) as indicated by the manufacturer.

    Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

    Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: Stimulation with glutamate is associated with the S-nitrosylation of CALHM1 in primary cultures of astrocytes. A Analysis performed through Proximity Ligation Assay (PLA) of the spatial association of eNOS or nNOS with CALHM1. Note that CALHM1 is associated with eNOS, but not with nNOS. A control in which primary antibodies were omitted (negative control) is also shown. B Representative Western blots (left) and densitometric analysis (right) of the changes in the levels of CALHM1 S-nitrosylation (CALHM1 S-NO) detected by biotin switch in primary cultures of astrocytes 3 min after the stimulation with 10 µM glutamate (Glut), 3 µM SNAP or the vehicle of glutamate (Vh). In addition, the effect of the inhibition of NO production with 100 µM N ω -nitro-L-arginine (L-NA) on the glutamate-elicited increase in CALHM1 S-NO is also shown. Variations in the level of CALHM1 S-NO are expressed in arbitrary units (A.U.). C Representative Western blot (WB) of CALHM1 S-nitrosylation using an anti-S-Nitroso-Cysteine antibody in astrocytes samples previously submitted to CALHM1 immunoprecipitation (IP). *P < 0.05 vs Vehicle by unpaired Student’s t-test

    Article Snippet: The sections were blocked with 0.5% BSA in PBS, incubated with a rabbit polyclonal primary antibody anti-CALHM1 (Alomone Labs, Israel) and a mouse monoclonal anti-GFAP (Sigma Aldrich, USA), and then, with an Alexa-568-labeled goat anti-rabbit secondary antibody and an Alexa-488-labeled goat anti-mouse secondary antibody (Invitrogen Molecular Probes, USA) using the Signal Enhancer HIKARI (Nacalai Tesque, INC, Japan) as indicated by the manufacturer.

    Techniques: Proximity Ligation Assay, Negative Control, Western Blot, Inhibition, Immunoprecipitation

    NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: NO activates CALHM1 channels in a heterologous expression system. A Representative current traces before and after application of 10 μM SNAP in a non-injected oocytes (NI) or oocytes expressing CALHM1 obtained at 1.8 mM extracellular Ca 2+ . Three CALHM1 expressing oocytes were additionally treated with 50 µM ascorbic acid, 10 µM ODQ or 20 µM ruthenium red (RuR). Oocytes were clamped to − 80 mV, and square pulses from − 80 mV to + 40 mV (in 10 mV steps) were then applied for 2 s. At the end of each pulse, the membrane potential was returned to − 80 mV. Note that ODQ did not affect NO-induced CALHM1 currents. B Normalized currents were obtained from the ratio between recorded current after and before 10 µM SNAP treatment. C Normalized currents at 0 mV oocytes resting membrane potential before and after 10 µM SNAP stimulation. Comparisons between groups were made using two-way ANOVA plus Tukey post-hoc test, *P < 0.05 vs Non-Injected (NI). D Time course of tail current peaks after reaching current saturation during a depolarizing pulses from − 80 to 20 mV (yellow box). Tail current peaks were obtained in the absence and presence of SNAP, and during RuR application. E Representative Western blot showing the expression and NO-mediated S-nitrosylation of CALHM1 in oocytes. From left to right, the first two lanes correspond to Western blots in non-injected oocytes (NI) and oocytes expressing CALHM1, and the next two lanes show the Biotin Switch analysis of CALHM1 S-nitrosylation observed in oocytes expressing CALHM1 in control conditions (lane 3) and after the stimulation with SNAP (lane 4)

    Article Snippet: The sections were blocked with 0.5% BSA in PBS, incubated with a rabbit polyclonal primary antibody anti-CALHM1 (Alomone Labs, Israel) and a mouse monoclonal anti-GFAP (Sigma Aldrich, USA), and then, with an Alexa-568-labeled goat anti-rabbit secondary antibody and an Alexa-488-labeled goat anti-mouse secondary antibody (Invitrogen Molecular Probes, USA) using the Signal Enhancer HIKARI (Nacalai Tesque, INC, Japan) as indicated by the manufacturer.

    Techniques: Expressing, Injection, Membrane, Western Blot

    Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

    Journal: Biological Research

    Article Title: Control of astrocytic Ca 2+ signaling by nitric oxide-dependent S-nitrosylation of Ca 2+ homeostasis modulator 1 channels

    doi: 10.1186/s40659-024-00503-3

    Figure Lengend Snippet: Schematic model of the signaling events that mediate the increase in [Ca 2+ ] i initiated by metabotropic glutamate receptor (mGluR) activation in astrocytes. Glutamate released during an increase in neuronal activity can exit the synaptic cleft and activate receptors on astrocyte processes. The activation of astrocyte mGluRs leads to an initial increase in [Ca 2+ ] i by the release of Ca 2+ from the intracellular Ca 2+ stores through activation of an inositol (1,4,5)-triphosphate (IP 3 )-mediated pathway. This astrocytic Ca 2+ signal triggers an increment in nitric oxide (NO) production by the endothelial NO synthase isoform (eNOS), which, in turn, evokes the opening of CALHM1 channels by the S-nitrosylation of this protein. The activation of CALHM1 channels play a pivotal role in the response by providing a pathway for ATP release and the sequential activation of P2 receptors (P2R), leading to the opening of Cx43 hemichannels and Panx-1 channels. Finally, the Ca 2+ influx through these membrane channels contributes to amplify the intracellular Ca 2+ store-initiated Ca 2+ signaling. In addition, the increase in [Ca 2+ ] i can be coordinated through the propagation of an inter-astrocyte Ca 2+ signal via ATP release or directly by gap junction communication (GJ)

    Article Snippet: The sections were blocked with 0.5% BSA in PBS, incubated with a rabbit polyclonal primary antibody anti-CALHM1 (Alomone Labs, Israel) and a mouse monoclonal anti-GFAP (Sigma Aldrich, USA), and then, with an Alexa-568-labeled goat anti-rabbit secondary antibody and an Alexa-488-labeled goat anti-mouse secondary antibody (Invitrogen Molecular Probes, USA) using the Signal Enhancer HIKARI (Nacalai Tesque, INC, Japan) as indicated by the manufacturer.

    Techniques: Activation Assay, Activity Assay, Membrane

    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).

    Journal: Cell reports

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    doi: 10.1016/j.celrep.2024.113834

    Figure Lengend Snippet: (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).

    Article Snippet: The primary antibodies used were sheep- anti -tyrosine hydroxylase (TH) polyclonal (catalog #ab113; Abcam Inc., Cambridge, MA, USA) at 1:1000 for labeling of DA neurons and a rabbit anti-α3-GABA A R subunit (extracellular) polyclonal (catalog #AGA-003; Alomone Labs, Jerusalem, Israel) at 1:200 for labeling GABA A Rs.

    Techniques: Confocal Microscopy, Labeling, Membrane, Immunolabeling, Expressing

    (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).

    Journal: Cell reports

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    doi: 10.1016/j.celrep.2024.113834

    Figure Lengend Snippet: (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).

    Article Snippet: The primary antibodies used were sheep- anti -tyrosine hydroxylase (TH) polyclonal (catalog #ab113; Abcam Inc., Cambridge, MA, USA) at 1:1000 for labeling of DA neurons and a rabbit anti-α3-GABA A R subunit (extracellular) polyclonal (catalog #AGA-003; Alomone Labs, Jerusalem, Israel) at 1:200 for labeling GABA A Rs.

    Techniques: Inhibition

    Journal: Cell reports

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    doi: 10.1016/j.celrep.2024.113834

    Figure Lengend Snippet:

    Article Snippet: The primary antibodies used were sheep- anti -tyrosine hydroxylase (TH) polyclonal (catalog #ab113; Abcam Inc., Cambridge, MA, USA) at 1:1000 for labeling of DA neurons and a rabbit anti-α3-GABA A R subunit (extracellular) polyclonal (catalog #AGA-003; Alomone Labs, Jerusalem, Israel) at 1:200 for labeling GABA A Rs.

    Techniques: Plasmid Preparation, Electron Microscopy, Blocking Assay, Virus, Recombinant, Saline, Microscopy, Software, Imaging

    (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).

    Journal: Cell reports

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    doi: 10.1016/j.celrep.2024.113834

    Figure Lengend Snippet: (A) Top: immunofluorescent images obtained with confocal microscopy showing co-localization of α3-GABA A R subunits with TH-labeled DA axons in dStr (left) and NAc core (right). White arrows indicate where co-localization is seen. Images are from a single plane of a z stack captured with a 60× Nikon oil-immersion objective (1.4 NA). Scale bar, 5 μm. Bottom: expanded views of a region of interest in the dStr at different levels in the z stack, demonstrating localization of α3-puncta in DA axonal profiles. Scale bar, 5 μm. (B) Left: electron micrographs showing α3-GABA A R subunits co-localized with TH-labeled DA axons in dStr and NAc core. Top: TH axons in dStr were identified by the presence of more than one SIG particle within an axonal profile packed with vesicles (red arrows), while presence of α3-GABA A R subunits was identified by electron-dense diffuse DAB-horseradish peroxidase (HRP) labeling of vesicular and plasma membranes (asterisks). α3 subunits within TH axons (red overlay) are highlighted by yellow asterisks, whereas α3-GABA A R subunits in non-TH axons (green overlay) are highlighted by white asterisks; UT marks an unlabeled axon terminal (yellow overlay). The white arrow points to the plasma membrane of a non-TH profile, identifiable as a dendritic spine receiving excitatory synaptic input from a non-TH axon terminal (rightmost asterisk), presumably glutamatergic and α3 positive. Scale bar, 200 nm. Bottom: TH axons and α3-GABA A R subunits in NAc core were identified as above and shown at higher magnification to highlight immunolabeling of the plasma membrane of a dually labeled axon (yellow arrow) and of vesicle membranes (encircled by yellow asterisks). Blue overlay shows a TH axon without α3. Scale bar, 200 nm. Right: average data, quantifying the percentage of TH axons expressing α3 (upper graph) and α3 axons expressing TH (lower graph) in dStr and NAc (n = 3 mice; mean ± SEM; ns is not signficant). (C) Cartoon showing generation of Ai32;DAT-Cre mice with genetic ChR2 expression in DAT-containing neurons and images of fixed coronal brain sections confirming selective fluorescent labeling of eYFP-ChR2 expression in midbrain DA (TH) soma and striatal axons. Scale bars, 0.5 mm. (D) Right: average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R agonist muscimol (Mus, 10 μM); error bars omitted for clarity. Muscimol significantly decreased 1 p-evoked [DA] o in both the dStr (n = 10 mice) and NAc (n = 12 from 11 mice). ***p < 0.01 versus respective control (ratio paired t test). Left: graph showing individual data (mean ± SEM) for the percentage decrease of peak [DA] o by muscimol in males (blue circles) plus females (orange circles) in dStr versus NAc (p > 0.05, unpaired t test; n is not significant).

    Article Snippet: Rabbit anti-GABA A R α 3 (extracellular) polyclonal , Alomone Labs , Cat No: AGA-003; RRID: AB_2039866.

    Techniques: Confocal Microscopy, Labeling, Membrane, Immunolabeling, Expressing

    (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).

    Journal: Cell reports

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    doi: 10.1016/j.celrep.2024.113834

    Figure Lengend Snippet: (A) Average optically evoked single-pulse (1 p) [DA] o transients recorded in dStr and NAc in the absence or presence of the GABA A R blocker picrotoxin (PTX, 100 μM); error bars omitted for clarity. (B) PTX significantly increased peak [DA] o evoked by 1 p in males (blue circles) plus females (orange circles) in both the dStr (n = 13 from 11 mice) and NAc (n = 10 from 9 mice). ***p < 0.001 versus respective control (ratio paired t test), thereby revealing inhibition of DA release by ambient GABA acting at GABA A Rs. The effect of PTX did not differ between dStr and NAc (p > 0.05, unpaired t test). Data are mean ± SEM; ns is not significant. (C) Efficacy of PTX-induced enhancement of DA release was variable but not related to initial evoked [DA] o under control conditions in either dStr (simple linear regression with Pearson correlation: slope = 6.494, R 2 = 0.001, F = 0.1075(1,11); p = 0.7492, n = 13) or NAc (simple linear regression with Pearson correlation: slope = −24.85, R 2 = 0.034, F = 0.2835(1,8); p = 0.6089, n = 10).

    Article Snippet: Rabbit anti-GABA A R α 3 (extracellular) polyclonal , Alomone Labs , Cat No: AGA-003; RRID: AB_2039866.

    Techniques: Inhibition

    Journal: Cell reports

    Article Title: GABA co-released from striatal dopamine axons dampens phasic dopamine release through autoregulatory GABA A receptors

    doi: 10.1016/j.celrep.2024.113834

    Figure Lengend Snippet:

    Article Snippet: Rabbit anti-GABA A R α 3 (extracellular) polyclonal , Alomone Labs , Cat No: AGA-003; RRID: AB_2039866.

    Techniques: Plasmid Preparation, Electron Microscopy, Blocking Assay, Virus, Recombinant, Saline, Microscopy, Software, Imaging

    a Single-molecule localization microscopy (SMLM) map showing Ca V 1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca V 1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c , d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in ( b , d ) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Journal: Nature Communications

    Article Title: BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

    doi: 10.1038/s41467-024-47847-8

    Figure Lengend Snippet: a Single-molecule localization microscopy (SMLM) map showing Ca V 1.2 channel localization and distribution in the t-tubules of young and old ventricular myocytes with or without ISO stimulation. Yellow boxes indicate the location of the regions of interest magnified in the top right of each image. b Dot-plots showing mean Ca V 1.2 channel cluster areas in young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 4, n = 11; ISO: N = 4, n = 10) myocytes. c , d show the same for RyR2 in young (control: N = 3, n = 15; ISO: N = 3, n = 15) and old (control: N = 3, n = 12; ISO: N = 3, n = 11) myocytes. Statistical analyses on data summarized in ( b , d ) were performed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in b and d are from NIA young. Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Article Snippet: Cells were incubated overnight at 4 °C in rabbit polyclonal IgG anti-Ca V 1.2 (CACNA1C, ACC-003, Alomone Labs, Jerusalem, Israel; 1:300 dilution) or mouse monoclonal IgG1 anti-RyR2 (C3-33, MA3-916, Invitrogen, Waltham, MA, USA; 1:50 dilution) in blocking buffer.

    Techniques: Microscopy, Comparison

    a Airyscan super-resolution images of Ca V 1.2 (green) and EEA1 (magenta) immunostained myocytes with and without ISO. Bottom: Binary colocalization maps show pixels in which Ca V 1.2 and EEA1 completely overlapped. b dot-plots summarizing % colocalization between EEA1 and Ca V 1.2 young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 14) myocytes, and c EEA1-positive endosome areas in young (control: N = 3, n = 15; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 13) myocytes. Data were analyzed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b , c ) are from JAX young. Note no significant differences in EEA1/Ca V 1.2 colocalization, responsivity to ISO, or endosome size was detected when young JAX and young NIA myocytes were compared (Supplementary Fig.  ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Journal: Nature Communications

    Article Title: BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

    doi: 10.1038/s41467-024-47847-8

    Figure Lengend Snippet: a Airyscan super-resolution images of Ca V 1.2 (green) and EEA1 (magenta) immunostained myocytes with and without ISO. Bottom: Binary colocalization maps show pixels in which Ca V 1.2 and EEA1 completely overlapped. b dot-plots summarizing % colocalization between EEA1 and Ca V 1.2 young (control: N = 3, n = 16; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 14) myocytes, and c EEA1-positive endosome areas in young (control: N = 3, n = 15; ISO: N = 3, n = 16) and old (control: N = 3, n = 14; ISO: N = 3, n = 13) myocytes. Data were analyzed using two-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b , c ) are from JAX young. Note no significant differences in EEA1/Ca V 1.2 colocalization, responsivity to ISO, or endosome size was detected when young JAX and young NIA myocytes were compared (Supplementary Fig. ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Article Snippet: Cells were incubated overnight at 4 °C in rabbit polyclonal IgG anti-Ca V 1.2 (CACNA1C, ACC-003, Alomone Labs, Jerusalem, Israel; 1:300 dilution) or mouse monoclonal IgG1 anti-RyR2 (C3-33, MA3-916, Invitrogen, Waltham, MA, USA; 1:50 dilution) in blocking buffer.

    Techniques: Comparison

    a Representative whole-cell currents from shRNA-scrmb and shRNA-mBIN1 myocytes before and during ISO application. b fold change in peak I Ca with ISO in young, old, shRNA-scrmb ( N = 3, n = 7), and shRNA-mBIN1 ( N = 3, n = 9) myocytes. c Representative Ca 2+ transients recorded from old shRNA-scrmb and shRNA-mBIN1 myocytes before and after ISO.  Fold increase after ISO from young, old, shRNA-scrmb ( N = 3, n = 14), and shRNA-mBIN1 ( N = 5, n = 18) myocytes. e SMLM localization maps showing Ca V 1.2 channel localization on t-tubules of myocytes from old shRNA-scrmb and shRNA-mBIN1, with or without ISO stimulation. Regions of interest are highlighted by yellow boxes. f Fold change in mean Ca V 1.2 channel cluster area with ISO in the young, old, old shRNA-scrmb (control: N = 3, n = 14; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 12; ISO: N = 3, n = 11). g , h show the same layout for RyR2 immunostained old shRNA-scrmb (control: N = 3, n = 12; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 9; ISO: N = 3, n = 8). Old and young data points in ( b ,  , f , h ) are reproduced from data in Figs.  b, h, and  b,  respectively. Statistical analysis was performed on data in ( b ,  , f , h ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b ) are pooled from NIA young and JAX young myocytes, data in (  , f , h ) are from NIA young myocytes. Note there was no significant difference in I Ca , Ca V 1.2, and RyR2 cluster areas when JAX and NIA young mice were compared (see Supplementary Fig.  ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Journal: Nature Communications

    Article Title: BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

    doi: 10.1038/s41467-024-47847-8

    Figure Lengend Snippet: a Representative whole-cell currents from shRNA-scrmb and shRNA-mBIN1 myocytes before and during ISO application. b fold change in peak I Ca with ISO in young, old, shRNA-scrmb ( N = 3, n = 7), and shRNA-mBIN1 ( N = 3, n = 9) myocytes. c Representative Ca 2+ transients recorded from old shRNA-scrmb and shRNA-mBIN1 myocytes before and after ISO. Fold increase after ISO from young, old, shRNA-scrmb ( N = 3, n = 14), and shRNA-mBIN1 ( N = 5, n = 18) myocytes. e SMLM localization maps showing Ca V 1.2 channel localization on t-tubules of myocytes from old shRNA-scrmb and shRNA-mBIN1, with or without ISO stimulation. Regions of interest are highlighted by yellow boxes. f Fold change in mean Ca V 1.2 channel cluster area with ISO in the young, old, old shRNA-scrmb (control: N = 3, n = 14; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 12; ISO: N = 3, n = 11). g , h show the same layout for RyR2 immunostained old shRNA-scrmb (control: N = 3, n = 12; ISO: N = 3, n = 13) and shRNA-mBIN1 myocytes (control: N = 3, n = 9; ISO: N = 3, n = 8). Old and young data points in ( b , , f , h ) are reproduced from data in Figs. b, h, and b, respectively. Statistical analysis was performed on data in ( b , , f , h ) using one-way ANOVAs with multiple comparison post-hoc tests. Young data in ( b ) are pooled from NIA young and JAX young myocytes, data in ( , f , h ) are from NIA young myocytes. Note there was no significant difference in I Ca , Ca V 1.2, and RyR2 cluster areas when JAX and NIA young mice were compared (see Supplementary Fig. ). Data are presented as mean ± SEM. Source data are provided in the Source Data file.

    Article Snippet: Cells were incubated overnight at 4 °C in rabbit polyclonal IgG anti-Ca V 1.2 (CACNA1C, ACC-003, Alomone Labs, Jerusalem, Israel; 1:300 dilution) or mouse monoclonal IgG1 anti-RyR2 (C3-33, MA3-916, Invitrogen, Waltham, MA, USA; 1:50 dilution) in blocking buffer.

    Techniques: shRNA, Comparison

    The main findings of our study graphically illustrated and summarized. Top : In healthy young cells, Ca V 1.2 channels undergo endosomal recycling, where channels on endosomes are either marked for degradation through the late endosome pathway or are recycled to the sarcolemma through the fast and slow recycling pathways. Following β -adrenergic receptor ( β -AR) stimulation, a pool of channels localized to endosomes are mobilized to the membrane, resulting in larger Ca V 1.2 clusters along t-tubules. Across the dyad, RyR2 clusters on the sarcoplasmic reticulum also increase following βAR stimulation ensuring efficient Ca 2+ -induced Ca 2+ -release. This increase in cytosolic Ca 2+ , along with increased phosphorylation of cardiac Troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) within the sarcomere, allows for enhanced contractility under acute stress to cope with elevated hemodynamic and metabolic demands. Bottom : In aging, Bridging Integrator 1 (BIN1) protein levels are increased, accompanied by a swelling of endosomes and subsequent dysregulation of endosomal trafficking of Ca V 1.2. Ca V 1.2 and RyR2 channels are basally super-clustered at the dyads and lose β -AR responsivity. Reduced phosphorylation of cTnI and cMyBP-C result in systolic and diastolic dysfunction. BIN1 knockdown in aging recovers RyR2 clustering plasticity and Ca 2+ transient responsivity to β- AR stimulation. Phosphorylation of cMyBP-C is basally restored, and contractility is recovered to youthful levels. Thus, BIN1 knockdown rejuvenates the aging heart. Created with BioRender.com.

    Journal: Nature Communications

    Article Title: BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

    doi: 10.1038/s41467-024-47847-8

    Figure Lengend Snippet: The main findings of our study graphically illustrated and summarized. Top : In healthy young cells, Ca V 1.2 channels undergo endosomal recycling, where channels on endosomes are either marked for degradation through the late endosome pathway or are recycled to the sarcolemma through the fast and slow recycling pathways. Following β -adrenergic receptor ( β -AR) stimulation, a pool of channels localized to endosomes are mobilized to the membrane, resulting in larger Ca V 1.2 clusters along t-tubules. Across the dyad, RyR2 clusters on the sarcoplasmic reticulum also increase following βAR stimulation ensuring efficient Ca 2+ -induced Ca 2+ -release. This increase in cytosolic Ca 2+ , along with increased phosphorylation of cardiac Troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C) within the sarcomere, allows for enhanced contractility under acute stress to cope with elevated hemodynamic and metabolic demands. Bottom : In aging, Bridging Integrator 1 (BIN1) protein levels are increased, accompanied by a swelling of endosomes and subsequent dysregulation of endosomal trafficking of Ca V 1.2. Ca V 1.2 and RyR2 channels are basally super-clustered at the dyads and lose β -AR responsivity. Reduced phosphorylation of cTnI and cMyBP-C result in systolic and diastolic dysfunction. BIN1 knockdown in aging recovers RyR2 clustering plasticity and Ca 2+ transient responsivity to β- AR stimulation. Phosphorylation of cMyBP-C is basally restored, and contractility is recovered to youthful levels. Thus, BIN1 knockdown rejuvenates the aging heart. Created with BioRender.com.

    Article Snippet: Cells were incubated overnight at 4 °C in rabbit polyclonal IgG anti-Ca V 1.2 (CACNA1C, ACC-003, Alomone Labs, Jerusalem, Israel; 1:300 dilution) or mouse monoclonal IgG1 anti-RyR2 (C3-33, MA3-916, Invitrogen, Waltham, MA, USA; 1:50 dilution) in blocking buffer.

    Techniques: Membrane, Binding Assay