kchip2 labelling Search Results


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  • 94
    Millipore anti calsenilin
    Increased Multi-Contact of Cones in the Bax null Retina and Associated with BC4 Density. a-c , BC4 organization and cone contacts in wild type (wt) retina. Dendritic boundaries outlined in yellow show tiling. c, Cones were contacted by a single BC4 (white circles) or multiple BC4s (red circles). d-f, BC4 dendrite territories, tiling and cone contacts in the Bax null retina. g, Cone contacts decrease in number as BC4 density increases in the wt (blue diamonds) and Bax null (red squares) retina. Each symbol represents the average number of cones contacted (2–9 cells total) for each image field that was analyzed. h, As density of BC4 increases, the percentage of multi-contacted cones increased for both wt (R 2 =0.6934) and Bax null (R 2 =0.6029) retinas with the percentage of cones contacted by multiple BC4s higher between neighboring BC4s in Bax null retinas. i, This trend remains when compared on a single cell basis: wt (R 2 =0.272) and Bax null (R 2 =0.189). j, The percent of cones that are multi- contacted between BC4s decreases as the dendritic arbor area of BC4s increases for both wt (R 2 =0.1634) and Bax null (R 2 =0.1941). k, As the number of total cones contacted increases for a single BC4, the percent of those cones that are multi-contact decreases in both the wt (R 2 =0.0928) and Bax null (R 2 =0.1478) retina. l , Multi-contact cones are on the borders of BC4 cell territories, while cones within larger BC4 territories are unable to be multi-contacted by more than one BC4 due to the BC4’s tiled organization. Abbreviations: wt= wild type; BC4= type 4 OFF bipolar cell; csen= <t>calsenilin;</t> PNA= peanut agglutinin. The scale bar in f is equal to 25 μm and is the equivalent in a - e. for a full record of test types and associated p-values.
    Anti Calsenilin, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Alomone Labs anti kchip2 antibody
    Protein expression and relative density of Kv4.3 and <t>KChiP2</t> subunits determined by Western blot and immunocytochemical techniques in sedentary and trained dog. Two bar diagrams on the left show the relative protein expression of Kv4.3 and KChiP2 subunits determined by Western blot in sedentary (n=12 dogs) and trained dog (n=12) left ventricular samples. Bottom panels indicates the representative Kv4.3 and KChIP2 bands and their corresponding loading control (GAPDH). Two bar diagrams on the right panels show the relative density of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling obtained from the sedentary (n=30 cells/6 dogs) and trained (n=30 cells/6 dogs) groups. Bottom panels on the right show original immunofluorescent images of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling. Figure 6–Source Data 1 Protein expression of Kv4.3 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 2 Protein expression of KChiP2 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 3 Relative density of Kv4.3 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 4 Relative density of KChiP2 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 5 Original, unedited membranes of western blots with the relevant bands clearly labelled. Figure 6–Source Data 6 Original files of the full raw unedited membranes of western blots.
    Anti Kchip2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kchip2 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    anti kchip2 antibody - by Bioz Stars, 2022-12
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    86
    Millipore mouse anti calsenilin
    Trnp1 marks ON type bipolar cells. Immunostaining of developing and adult retinas with Trnp1 ( green ) and cell type-specific markers. ( A – E ) Trnp1 costaining with Otx2 ( red ) and Pax6 ( gray ) at multiple ages. Otx2 is cropped and Pax6 shown only in the insets for clarity. At P0 ( A ) and P5 ( B ), no Trnp1 immunostaining is detected in the retina. ( C ) Starting at P7, Trnp1 nuclear staining is seen in the INL, where it overlaps completely with Otx2 ( arrows , insets ). The same pattern of Trnp1 expression is seen at P10 ( D ) and in adult ( E ) sections. Pax6+ amacrine cells in the ONL ( arrowheads , insets ) do not coexpress Trnp1 at any age. ( F – K ) Adult sections stained with Trnp1 and bipolar subtype specific markers ( red / gray ). ( F ) Cells that are Trnp1+ coexpress Isl1/2 ( red , arrows , insets ), which marks ON type bipolar cells in the retina. Starburst amacrines labeled by Isl1/2 ( arrowheads ) do not express Trnp1. ( G – G'' ) A section showing Trnp1, Scgn ( gray ) and PKCα ( red ) costaining. A subset of Trnp1+ cells coexpresses Scgn ( arrowheads , insets ) or PKCα ( arrows , insets ). Nearly all of the PKCα+ rod bipolar cells express Trnp1 (G''), but only a fraction of Scgn+ cone bipolars are Trnp1+ ( G' ). ( H ) Type 2 cone OFF bipolar cells marked by Bhlhb5 staining ( arrowheads , insets ) do not coexpress Trnp1. Bhlhb5+ amacrine cells are marked with an “a”. ( I ) <t>Calsenilin-positive</t> type 4 cone OFF bipolar cells ( arrowheads , insets ) do not coexpress Trnp1. Scale bars : ( A – E , G – H ) 25 μm for panels and 10 μm for insets; ( F ) 100 μm and 10 μm for insets; ( I ) 50 μm and 10 μm for the insets. ( J ) Quantification of Trnp1 staining in the adult wild-type retina. The left panel shows the fraction of Trnp1+ cells that coexpress a cell-type specific marker. The right panel shows what percentage of a given population of cells expresses Trnp1. Error bars represent SD.
    Mouse Anti Calsenilin, supplied by Millipore, 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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    94
    Alomone Labs kchip2 labelling
    Increasing I to,f density with bicistronic <t>Kv4.3/KChIP2.1</t> expression affects cardiomyocyte AP waveform. Panel A shows APs recorded from 2 day cultured (untransduced black), and transduced myocytes with increasing I to,f current densities at a cycle length of 1 s. Introduction of I to,f (lower panel traces show exemplar records for pulses to −30, +10 and + 40 mV) resulted in a prominent AP phase 1 in all transduced myocytes. As I to,f density increased, there was a progressive change in AP morphology that caused the AP to resemble that recorded in other species. B. Increasing density of I to,f (at +40 mV) decreased APD20. APD90 (right panel) decreased approximately exponentially (solid line) with increasing I to,f except when I to,f was between ~12 and ~ 17 pA/pF (deviation highlighted as dashed line) where emergence of a marked ‘spike-and-dome’ morphology developed (as shown center right in A). All trend curves drawn by eye. C. Representative traces of I to,f recorded in untransduced (Control) iPSC-CMs and iPSC-CMs expressing bicistronic Kv4.3-mCherry/KChIP2.1 under a cTnT promoter. After a brief step to −40 mV to inactivate Na + channels, subsequent membrane depolarizations to −30 mV, −10 mV, +10 mV, +30 mV and + 50 mV were used to elicit I to in control and transduced iPSC-CMs. D. Three examplar AP waveforms of iPSC-CMs without (black traces) or with expressed I to,f currents (green lower traces). I to,f densities in the exemplar transduced iPSC-CMs were 5–10 pA/pF at +40 mV. APs were elicited at a frequency of 1 Hz. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Kchip2 Labelling, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Increased Multi-Contact of Cones in the Bax null Retina and Associated with BC4 Density. a-c , BC4 organization and cone contacts in wild type (wt) retina. Dendritic boundaries outlined in yellow show tiling. c, Cones were contacted by a single BC4 (white circles) or multiple BC4s (red circles). d-f, BC4 dendrite territories, tiling and cone contacts in the Bax null retina. g, Cone contacts decrease in number as BC4 density increases in the wt (blue diamonds) and Bax null (red squares) retina. Each symbol represents the average number of cones contacted (2–9 cells total) for each image field that was analyzed. h, As density of BC4 increases, the percentage of multi-contacted cones increased for both wt (R 2 =0.6934) and Bax null (R 2 =0.6029) retinas with the percentage of cones contacted by multiple BC4s higher between neighboring BC4s in Bax null retinas. i, This trend remains when compared on a single cell basis: wt (R 2 =0.272) and Bax null (R 2 =0.189). j, The percent of cones that are multi- contacted between BC4s decreases as the dendritic arbor area of BC4s increases for both wt (R 2 =0.1634) and Bax null (R 2 =0.1941). k, As the number of total cones contacted increases for a single BC4, the percent of those cones that are multi-contact decreases in both the wt (R 2 =0.0928) and Bax null (R 2 =0.1478) retina. l , Multi-contact cones are on the borders of BC4 cell territories, while cones within larger BC4 territories are unable to be multi-contacted by more than one BC4 due to the BC4’s tiled organization. Abbreviations: wt= wild type; BC4= type 4 OFF bipolar cell; csen= calsenilin; PNA= peanut agglutinin. The scale bar in f is equal to 25 μm and is the equivalent in a - e. for a full record of test types and associated p-values.

    Journal: The Journal of comparative neurology

    Article Title: Increased density and age-related sharing of synapses at the cone to OFF bipolar cell synapse in the mouse retina

    doi: 10.1002/cne.24810

    Figure Lengend Snippet: Increased Multi-Contact of Cones in the Bax null Retina and Associated with BC4 Density. a-c , BC4 organization and cone contacts in wild type (wt) retina. Dendritic boundaries outlined in yellow show tiling. c, Cones were contacted by a single BC4 (white circles) or multiple BC4s (red circles). d-f, BC4 dendrite territories, tiling and cone contacts in the Bax null retina. g, Cone contacts decrease in number as BC4 density increases in the wt (blue diamonds) and Bax null (red squares) retina. Each symbol represents the average number of cones contacted (2–9 cells total) for each image field that was analyzed. h, As density of BC4 increases, the percentage of multi-contacted cones increased for both wt (R 2 =0.6934) and Bax null (R 2 =0.6029) retinas with the percentage of cones contacted by multiple BC4s higher between neighboring BC4s in Bax null retinas. i, This trend remains when compared on a single cell basis: wt (R 2 =0.272) and Bax null (R 2 =0.189). j, The percent of cones that are multi- contacted between BC4s decreases as the dendritic arbor area of BC4s increases for both wt (R 2 =0.1634) and Bax null (R 2 =0.1941). k, As the number of total cones contacted increases for a single BC4, the percent of those cones that are multi-contact decreases in both the wt (R 2 =0.0928) and Bax null (R 2 =0.1478) retina. l , Multi-contact cones are on the borders of BC4 cell territories, while cones within larger BC4 territories are unable to be multi-contacted by more than one BC4 due to the BC4’s tiled organization. Abbreviations: wt= wild type; BC4= type 4 OFF bipolar cell; csen= calsenilin; PNA= peanut agglutinin. The scale bar in f is equal to 25 μm and is the equivalent in a - e. for a full record of test types and associated p-values.

    Article Snippet: The following antibody and lectins were utilized in this study: Rabbit anti-cone arrestin (cones; Millipore; AB15282; 1:1000: RRID:AB_1163387), mouse (IgG1) anti-calsenilin (type 4 cone bipolar cells; Millipore; 05–756; 1:500: RRID:AB_309969), mouse (IgG1) anti-Goa (ON bipolar cells; Millipore; MAB3073; 1:1000: RRID:AB_94671), Peanut agglutinin (PNA) (cone pedicles; Molecular Probes; ; 1:1000: RRID:AB_2315178), rabbit anti-PkaRIIβ (type 3b bipolar cells; BD biosciences 610625; 1:500: RRID:AB_397957), rabbit anti-PKCα (rod bipolar cells; Santa Cruz Biotechnology; sc-10800; 1:500: RRID:AB_2168560).

    Techniques:

    Dendrite Tip and Projection Based on Contact Patterns. a and b, BC4s labeled with ai9, using the calsenilin antibody to confirm identity (not shown) and cones labeled with PNA. Asterisks in a and b represent singly contacted ( a ) and multi-contacted cones ( b ). c , The number of BC4 dendrite tips that contact a cone increase when cones are multi-contacted and this was not dependent on density. d, The overlap between PNA and ai9 was increased for multi-contacted cones compared to controls. Overlap with PNA increases when cell density decreases. Statistical comparisons were performed using ANOVA tests coupled with Dunn post hoc tests. Abbreviations: LD: low density, HD: high density, SC: single contact, MC: multi-contact. Scale bar in b is equal to 10 μm and is the same for a and b for a full record of test types and associated p-values.

    Journal: The Journal of comparative neurology

    Article Title: Increased density and age-related sharing of synapses at the cone to OFF bipolar cell synapse in the mouse retina

    doi: 10.1002/cne.24810

    Figure Lengend Snippet: Dendrite Tip and Projection Based on Contact Patterns. a and b, BC4s labeled with ai9, using the calsenilin antibody to confirm identity (not shown) and cones labeled with PNA. Asterisks in a and b represent singly contacted ( a ) and multi-contacted cones ( b ). c , The number of BC4 dendrite tips that contact a cone increase when cones are multi-contacted and this was not dependent on density. d, The overlap between PNA and ai9 was increased for multi-contacted cones compared to controls. Overlap with PNA increases when cell density decreases. Statistical comparisons were performed using ANOVA tests coupled with Dunn post hoc tests. Abbreviations: LD: low density, HD: high density, SC: single contact, MC: multi-contact. Scale bar in b is equal to 10 μm and is the same for a and b for a full record of test types and associated p-values.

    Article Snippet: The following antibody and lectins were utilized in this study: Rabbit anti-cone arrestin (cones; Millipore; AB15282; 1:1000: RRID:AB_1163387), mouse (IgG1) anti-calsenilin (type 4 cone bipolar cells; Millipore; 05–756; 1:500: RRID:AB_309969), mouse (IgG1) anti-Goa (ON bipolar cells; Millipore; MAB3073; 1:1000: RRID:AB_94671), Peanut agglutinin (PNA) (cone pedicles; Molecular Probes; ; 1:1000: RRID:AB_2315178), rabbit anti-PkaRIIβ (type 3b bipolar cells; BD biosciences 610625; 1:500: RRID:AB_397957), rabbit anti-PKCα (rod bipolar cells; Santa Cruz Biotechnology; sc-10800; 1:500: RRID:AB_2168560).

    Techniques: Labeling

    BC4 Contact Patterns at Cone Photoreceptors. a-d, BC4 cells labeled with antibodies to calsenilin (csen). The density of BC4s varies depending on genotype and retina region ( a and b , central retina; c and d , peripheral retina). e , Significantly (*) more BC4s were observed in the Bax null retina compared to wild type (wt) (C: central, M: middle, P: peripheral. e - g , Lines under the columns represent comparisons within genotype regions with asterisks (*) denoting significant differences. The top three open bars represent comparisons within the wild type retina and the bottom three solid bars represent comparisons within the Bax null retina. f , Consistent with previous findings, cone density is not significantly affected in Bax null retina compared to wt. g , For all three regions, the ratio of BC4/cone is significantly higher in Bax null retina compared to wt. h , Retinas were labeled with a transgene encoding red fluorescent proteins (ai9) and antibodies to calsenilin. Ai9 mostly label BC4s but the calsenilin antibody (*) was necessary to confirm cell type because ai9 was observed in some other BCs and a GFP transgene also used in the study was not observed in all BC4s. i , BC4 dendrites labeled with ai9 and GFP, and peanut agglutinin (PNA) to label cone terminals. j ). Individual BC4s were traced and polygonal dendritic arbor areas measured (yellow lines) in wt ( j and k ) and Bax null (l) retinas. m - o , BC4s labeled with ai9 and calsenilin, and cones labeled with PNA. n, Dendrite tracings of identified BC4s in m, and their contact with surrounding cones. Asterisks (*) denote multi-contacted cones. o, Dendrite tracings of BC4s from l . p, The percent of cones contacted by no BC4s (0I), one BC4 (SI) and multiple BC4s (MI) determined at postnatal days 15, three, five and 12 weeks, and five months. An increase in the percent of cones contacted by multiple BC4s increased over time. An ANOVA test identified significant differences across ages; a post hoc test of significant difference is annotated by an asterisk at each point of difference between time points represented by a bar representing the ages compared. Abbreviations: wt= wild type; BC4= type 4 OFF bipolar cell; C= central retina; M=middle; P=peripheral; csen= calsenilin; PNA= peanut agglutinin. The scale bar in d is equal to 50 μm and is equivalent for a - d . The scale bar in h is equal to 25 μm. The scale bar in k is equal to 25 μm and is the same for i - k . The scale bar in l is equal to 10 μm and is equivalent in l - o . Asterisks in e - g and p for a full record of test types and associated p-values.

    Journal: The Journal of comparative neurology

    Article Title: Increased density and age-related sharing of synapses at the cone to OFF bipolar cell synapse in the mouse retina

    doi: 10.1002/cne.24810

    Figure Lengend Snippet: BC4 Contact Patterns at Cone Photoreceptors. a-d, BC4 cells labeled with antibodies to calsenilin (csen). The density of BC4s varies depending on genotype and retina region ( a and b , central retina; c and d , peripheral retina). e , Significantly (*) more BC4s were observed in the Bax null retina compared to wild type (wt) (C: central, M: middle, P: peripheral. e - g , Lines under the columns represent comparisons within genotype regions with asterisks (*) denoting significant differences. The top three open bars represent comparisons within the wild type retina and the bottom three solid bars represent comparisons within the Bax null retina. f , Consistent with previous findings, cone density is not significantly affected in Bax null retina compared to wt. g , For all three regions, the ratio of BC4/cone is significantly higher in Bax null retina compared to wt. h , Retinas were labeled with a transgene encoding red fluorescent proteins (ai9) and antibodies to calsenilin. Ai9 mostly label BC4s but the calsenilin antibody (*) was necessary to confirm cell type because ai9 was observed in some other BCs and a GFP transgene also used in the study was not observed in all BC4s. i , BC4 dendrites labeled with ai9 and GFP, and peanut agglutinin (PNA) to label cone terminals. j ). Individual BC4s were traced and polygonal dendritic arbor areas measured (yellow lines) in wt ( j and k ) and Bax null (l) retinas. m - o , BC4s labeled with ai9 and calsenilin, and cones labeled with PNA. n, Dendrite tracings of identified BC4s in m, and their contact with surrounding cones. Asterisks (*) denote multi-contacted cones. o, Dendrite tracings of BC4s from l . p, The percent of cones contacted by no BC4s (0I), one BC4 (SI) and multiple BC4s (MI) determined at postnatal days 15, three, five and 12 weeks, and five months. An increase in the percent of cones contacted by multiple BC4s increased over time. An ANOVA test identified significant differences across ages; a post hoc test of significant difference is annotated by an asterisk at each point of difference between time points represented by a bar representing the ages compared. Abbreviations: wt= wild type; BC4= type 4 OFF bipolar cell; C= central retina; M=middle; P=peripheral; csen= calsenilin; PNA= peanut agglutinin. The scale bar in d is equal to 50 μm and is equivalent for a - d . The scale bar in h is equal to 25 μm. The scale bar in k is equal to 25 μm and is the same for i - k . The scale bar in l is equal to 10 μm and is equivalent in l - o . Asterisks in e - g and p for a full record of test types and associated p-values.

    Article Snippet: The following antibody and lectins were utilized in this study: Rabbit anti-cone arrestin (cones; Millipore; AB15282; 1:1000: RRID:AB_1163387), mouse (IgG1) anti-calsenilin (type 4 cone bipolar cells; Millipore; 05–756; 1:500: RRID:AB_309969), mouse (IgG1) anti-Goa (ON bipolar cells; Millipore; MAB3073; 1:1000: RRID:AB_94671), Peanut agglutinin (PNA) (cone pedicles; Molecular Probes; ; 1:1000: RRID:AB_2315178), rabbit anti-PkaRIIβ (type 3b bipolar cells; BD biosciences 610625; 1:500: RRID:AB_397957), rabbit anti-PKCα (rod bipolar cells; Santa Cruz Biotechnology; sc-10800; 1:500: RRID:AB_2168560).

    Techniques: Labeling

    Protein expression and relative density of Kv4.3 and KChiP2 subunits determined by Western blot and immunocytochemical techniques in sedentary and trained dog. Two bar diagrams on the left show the relative protein expression of Kv4.3 and KChiP2 subunits determined by Western blot in sedentary (n=12 dogs) and trained dog (n=12) left ventricular samples. Bottom panels indicates the representative Kv4.3 and KChIP2 bands and their corresponding loading control (GAPDH). Two bar diagrams on the right panels show the relative density of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling obtained from the sedentary (n=30 cells/6 dogs) and trained (n=30 cells/6 dogs) groups. Bottom panels on the right show original immunofluorescent images of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling. Figure 6–Source Data 1 Protein expression of Kv4.3 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 2 Protein expression of KChiP2 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 3 Relative density of Kv4.3 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 4 Relative density of KChiP2 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 5 Original, unedited membranes of western blots with the relevant bands clearly labelled. Figure 6–Source Data 6 Original files of the full raw unedited membranes of western blots.

    Journal: bioRxiv

    Article Title: Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibilty in a canine model of elite exercise

    doi: 10.1101/2022.07.13.499876

    Figure Lengend Snippet: Protein expression and relative density of Kv4.3 and KChiP2 subunits determined by Western blot and immunocytochemical techniques in sedentary and trained dog. Two bar diagrams on the left show the relative protein expression of Kv4.3 and KChiP2 subunits determined by Western blot in sedentary (n=12 dogs) and trained dog (n=12) left ventricular samples. Bottom panels indicates the representative Kv4.3 and KChIP2 bands and their corresponding loading control (GAPDH). Two bar diagrams on the right panels show the relative density of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling obtained from the sedentary (n=30 cells/6 dogs) and trained (n=30 cells/6 dogs) groups. Bottom panels on the right show original immunofluorescent images of dog cardiomyocytes with Kv4.3 and KChiP2 immunolabelling. Figure 6–Source Data 1 Protein expression of Kv4.3 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 2 Protein expression of KChiP2 subunit determined by Western blot technique in sedentary and trained dogs. Figure 6–Source Data 3 Relative density of Kv4.3 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 4 Relative density of KChiP2 subunit determined by immunocytochemical technique in sedentary and trained dogs. Figure 6–Source Data 5 Original, unedited membranes of western blots with the relevant bands clearly labelled. Figure 6–Source Data 6 Original files of the full raw unedited membranes of western blots.

    Article Snippet: After the incubation period, cells were labelled overnight at 4°C with anti-KChIP2 (Alomone, #APC-142, RRID:AB_2756744), anti-Kv4.3 (Alomone, #APC-017, RRID:AB_2040178), anti-HCN1 (Alomone, #APC-056, RRID:AB_2039900), anti-HCN2 (Alomone, #APC-030, RRID:AB_2313726) and anti-HCN4 (Alomone, #APC-052, RRID:AB_2039906) primary antibody diluted to 1:50.

    Techniques: Expressing, Western Blot

    Trnp1 marks ON type bipolar cells. Immunostaining of developing and adult retinas with Trnp1 ( green ) and cell type-specific markers. ( A – E ) Trnp1 costaining with Otx2 ( red ) and Pax6 ( gray ) at multiple ages. Otx2 is cropped and Pax6 shown only in the insets for clarity. At P0 ( A ) and P5 ( B ), no Trnp1 immunostaining is detected in the retina. ( C ) Starting at P7, Trnp1 nuclear staining is seen in the INL, where it overlaps completely with Otx2 ( arrows , insets ). The same pattern of Trnp1 expression is seen at P10 ( D ) and in adult ( E ) sections. Pax6+ amacrine cells in the ONL ( arrowheads , insets ) do not coexpress Trnp1 at any age. ( F – K ) Adult sections stained with Trnp1 and bipolar subtype specific markers ( red / gray ). ( F ) Cells that are Trnp1+ coexpress Isl1/2 ( red , arrows , insets ), which marks ON type bipolar cells in the retina. Starburst amacrines labeled by Isl1/2 ( arrowheads ) do not express Trnp1. ( G – G'' ) A section showing Trnp1, Scgn ( gray ) and PKCα ( red ) costaining. A subset of Trnp1+ cells coexpresses Scgn ( arrowheads , insets ) or PKCα ( arrows , insets ). Nearly all of the PKCα+ rod bipolar cells express Trnp1 (G''), but only a fraction of Scgn+ cone bipolars are Trnp1+ ( G' ). ( H ) Type 2 cone OFF bipolar cells marked by Bhlhb5 staining ( arrowheads , insets ) do not coexpress Trnp1. Bhlhb5+ amacrine cells are marked with an “a”. ( I ) Calsenilin-positive type 4 cone OFF bipolar cells ( arrowheads , insets ) do not coexpress Trnp1. Scale bars : ( A – E , G – H ) 25 μm for panels and 10 μm for insets; ( F ) 100 μm and 10 μm for insets; ( I ) 50 μm and 10 μm for the insets. ( J ) Quantification of Trnp1 staining in the adult wild-type retina. The left panel shows the fraction of Trnp1+ cells that coexpress a cell-type specific marker. The right panel shows what percentage of a given population of cells expresses Trnp1. Error bars represent SD.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina

    doi: 10.1167/iovs.16-19767

    Figure Lengend Snippet: Trnp1 marks ON type bipolar cells. Immunostaining of developing and adult retinas with Trnp1 ( green ) and cell type-specific markers. ( A – E ) Trnp1 costaining with Otx2 ( red ) and Pax6 ( gray ) at multiple ages. Otx2 is cropped and Pax6 shown only in the insets for clarity. At P0 ( A ) and P5 ( B ), no Trnp1 immunostaining is detected in the retina. ( C ) Starting at P7, Trnp1 nuclear staining is seen in the INL, where it overlaps completely with Otx2 ( arrows , insets ). The same pattern of Trnp1 expression is seen at P10 ( D ) and in adult ( E ) sections. Pax6+ amacrine cells in the ONL ( arrowheads , insets ) do not coexpress Trnp1 at any age. ( F – K ) Adult sections stained with Trnp1 and bipolar subtype specific markers ( red / gray ). ( F ) Cells that are Trnp1+ coexpress Isl1/2 ( red , arrows , insets ), which marks ON type bipolar cells in the retina. Starburst amacrines labeled by Isl1/2 ( arrowheads ) do not express Trnp1. ( G – G'' ) A section showing Trnp1, Scgn ( gray ) and PKCα ( red ) costaining. A subset of Trnp1+ cells coexpresses Scgn ( arrowheads , insets ) or PKCα ( arrows , insets ). Nearly all of the PKCα+ rod bipolar cells express Trnp1 (G''), but only a fraction of Scgn+ cone bipolars are Trnp1+ ( G' ). ( H ) Type 2 cone OFF bipolar cells marked by Bhlhb5 staining ( arrowheads , insets ) do not coexpress Trnp1. Bhlhb5+ amacrine cells are marked with an “a”. ( I ) Calsenilin-positive type 4 cone OFF bipolar cells ( arrowheads , insets ) do not coexpress Trnp1. Scale bars : ( A – E , G – H ) 25 μm for panels and 10 μm for insets; ( F ) 100 μm and 10 μm for insets; ( I ) 50 μm and 10 μm for the insets. ( J ) Quantification of Trnp1 staining in the adult wild-type retina. The left panel shows the fraction of Trnp1+ cells that coexpress a cell-type specific marker. The right panel shows what percentage of a given population of cells expresses Trnp1. Error bars represent SD.

    Article Snippet: Primary antibodies used were: mouse anti-Ap2α (1:250, clone 5E4; Developmental Studies Hybridoma Bank, Iowa City, IA, USA); chicken anti–β-galactosidase (β-gal; 1:2000, AB9361; Abcam, Cambridge, MA, USA); goat anti-Bhlhb5 (1:1000, sc-6045; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-Cabp5 (1:10, a gift from F. Haeseleer, University of Washington) ; mouse anti-Calretinin (1:750) (MAB1568, Milipore, Billerica, MA, USA); mouse anti-Calsenilin (1:2000, 05-756; Milipore); rabbit anti-GAD65/67 (1:500, AB1511; Milipore); goat anti-GlyT1 (1:2000, AB1770; Milipore); rabbit anti-HCN4 (1:500, APC-052; Alomone Labs Ltd., Jerusalem, Israel); mouse anti-Isl1/2 (1:250, clone 39.4D5; Developmental Studies Hybridoma Bank); goat anti-Otx2 (1:200, BAF1979; R & D Systems, Minneapolis, MN, USA); rabbit anti-Pax6 (1:500, 901301; BioLegend, Inc., San Diego, CA, USA); mouse anti-PKARIIβ (1:3000, 610625; BD Biosciences, San Jose, CA, USA); mouse anti-PKCα (1:250, P5704; Sigma-Aldrich Corp., St. Louis, MO, USA); rabbit anti-Scgn (1:5000, RD181120100; Biovendor LLC, Ashville, NC, USA); goat anti-Sox2 (1:100, sc17320; Santa Cruz Biotechnology); guinea pig anti-Trnp1 (1:200, a gift from M. Götz, Helmholtz Zentrum Muenchen) ; and rabbit anti-Vsx1 (1:250, a gift from E. Levine, Vanderbilt University).

    Techniques: Immunostaining, Staining, Expressing, Labeling, Marker

    Increasing I to,f density with bicistronic Kv4.3/KChIP2.1 expression affects cardiomyocyte AP waveform. Panel A shows APs recorded from 2 day cultured (untransduced black), and transduced myocytes with increasing I to,f current densities at a cycle length of 1 s. Introduction of I to,f (lower panel traces show exemplar records for pulses to −30, +10 and + 40 mV) resulted in a prominent AP phase 1 in all transduced myocytes. As I to,f density increased, there was a progressive change in AP morphology that caused the AP to resemble that recorded in other species. B. Increasing density of I to,f (at +40 mV) decreased APD20. APD90 (right panel) decreased approximately exponentially (solid line) with increasing I to,f except when I to,f was between ~12 and ~ 17 pA/pF (deviation highlighted as dashed line) where emergence of a marked ‘spike-and-dome’ morphology developed (as shown center right in A). All trend curves drawn by eye. C. Representative traces of I to,f recorded in untransduced (Control) iPSC-CMs and iPSC-CMs expressing bicistronic Kv4.3-mCherry/KChIP2.1 under a cTnT promoter. After a brief step to −40 mV to inactivate Na + channels, subsequent membrane depolarizations to −30 mV, −10 mV, +10 mV, +30 mV and + 50 mV were used to elicit I to in control and transduced iPSC-CMs. D. Three examplar AP waveforms of iPSC-CMs without (black traces) or with expressed I to,f currents (green lower traces). I to,f densities in the exemplar transduced iPSC-CMs were 5–10 pA/pF at +40 mV. APs were elicited at a frequency of 1 Hz. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

    doi: 10.1016/j.yjmcc.2021.11.004

    Figure Lengend Snippet: Increasing I to,f density with bicistronic Kv4.3/KChIP2.1 expression affects cardiomyocyte AP waveform. Panel A shows APs recorded from 2 day cultured (untransduced black), and transduced myocytes with increasing I to,f current densities at a cycle length of 1 s. Introduction of I to,f (lower panel traces show exemplar records for pulses to −30, +10 and + 40 mV) resulted in a prominent AP phase 1 in all transduced myocytes. As I to,f density increased, there was a progressive change in AP morphology that caused the AP to resemble that recorded in other species. B. Increasing density of I to,f (at +40 mV) decreased APD20. APD90 (right panel) decreased approximately exponentially (solid line) with increasing I to,f except when I to,f was between ~12 and ~ 17 pA/pF (deviation highlighted as dashed line) where emergence of a marked ‘spike-and-dome’ morphology developed (as shown center right in A). All trend curves drawn by eye. C. Representative traces of I to,f recorded in untransduced (Control) iPSC-CMs and iPSC-CMs expressing bicistronic Kv4.3-mCherry/KChIP2.1 under a cTnT promoter. After a brief step to −40 mV to inactivate Na + channels, subsequent membrane depolarizations to −30 mV, −10 mV, +10 mV, +30 mV and + 50 mV were used to elicit I to in control and transduced iPSC-CMs. D. Three examplar AP waveforms of iPSC-CMs without (black traces) or with expressed I to,f currents (green lower traces). I to,f densities in the exemplar transduced iPSC-CMs were 5–10 pA/pF at +40 mV. APs were elicited at a frequency of 1 Hz. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

    Techniques: Expressing, Cell Culture

    A. Plasmid vector for bicistronic for Kv4.3 and KChIP2.1 with amino acids shown in black (linkers in brown). A CMV or cardiac-specific troponin T promoter (cTnT) was used in different cell types. B. Western blot of KChIP2.1 expression by the bicistronic transgene in HEK293 cells. A single band corresponding to the estimated molecular weight of KChIP2.1 or KChIP2.1-Amcyan was detected in lysates from bicistronic transgene transfected cells but not from cells transfected with Kv4.3-mCherry alone. C Expression of Kv4.3-mCherry upstream of the P2A sequence was detected at an apparent molecular weight of ~80 kDa while expression of Kv4.3 alone was detected at ~60 kDa. These data are consistent with the expected weights of Kv4.3 (71 kDa) and mCherry (20 kDa), given a − 20% gel shift for K channel protein [ 61 ]. Additional higher molecular weight bands possibly reflect multimeric forms of Kv4.3. Lysates of cells transfected with untagged Kv4.3 and Amcyan-P2A-mCherry were used as positive and negative controls respectively and SYPRO® staining was used as internal loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

    doi: 10.1016/j.yjmcc.2021.11.004

    Figure Lengend Snippet: A. Plasmid vector for bicistronic for Kv4.3 and KChIP2.1 with amino acids shown in black (linkers in brown). A CMV or cardiac-specific troponin T promoter (cTnT) was used in different cell types. B. Western blot of KChIP2.1 expression by the bicistronic transgene in HEK293 cells. A single band corresponding to the estimated molecular weight of KChIP2.1 or KChIP2.1-Amcyan was detected in lysates from bicistronic transgene transfected cells but not from cells transfected with Kv4.3-mCherry alone. C Expression of Kv4.3-mCherry upstream of the P2A sequence was detected at an apparent molecular weight of ~80 kDa while expression of Kv4.3 alone was detected at ~60 kDa. These data are consistent with the expected weights of Kv4.3 (71 kDa) and mCherry (20 kDa), given a − 20% gel shift for K channel protein [ 61 ]. Additional higher molecular weight bands possibly reflect multimeric forms of Kv4.3. Lysates of cells transfected with untagged Kv4.3 and Amcyan-P2A-mCherry were used as positive and negative controls respectively and SYPRO® staining was used as internal loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

    Techniques: Plasmid Preparation, Western Blot, Expressing, Molecular Weight, Transfection, Sequencing, Electrophoretic Mobility Shift Assay, Staining

    Subcellular colocalization of Kv4.3 and KChIP2.1. A. Exemplar confocal images of living HEK293 cells expressing bicistronic Kv4.3-mCherry and KChIP2.1-AmCyan. B. Representative confocal images of a transduced rabbit ventricular cardiomyocytes expressing Kv4.3-mCherry and KChIP2 (see also Fig. S2A). Surface membranes were labelled with an anti-Cav3 antibody (top). C shows an enlarged view of the boxed regions in B and the fluorescence intensity profile from each label at bottom. D Co-localization between Kv4.3 and KChIP2.1 in different subcellular compartments (sarcolemma -SL; t-tubules TT; and remaining cytoplasm -Cyt.) measured by unbiased Pearson's correlation coefficient (n/ N = 8/3). Error bars show s.e.m.

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

    doi: 10.1016/j.yjmcc.2021.11.004

    Figure Lengend Snippet: Subcellular colocalization of Kv4.3 and KChIP2.1. A. Exemplar confocal images of living HEK293 cells expressing bicistronic Kv4.3-mCherry and KChIP2.1-AmCyan. B. Representative confocal images of a transduced rabbit ventricular cardiomyocytes expressing Kv4.3-mCherry and KChIP2 (see also Fig. S2A). Surface membranes were labelled with an anti-Cav3 antibody (top). C shows an enlarged view of the boxed regions in B and the fluorescence intensity profile from each label at bottom. D Co-localization between Kv4.3 and KChIP2.1 in different subcellular compartments (sarcolemma -SL; t-tubules TT; and remaining cytoplasm -Cyt.) measured by unbiased Pearson's correlation coefficient (n/ N = 8/3). Error bars show s.e.m.

    Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

    Techniques: Expressing, Fluorescence

    Biophysical properties of I to,f currents produced by Kv4.3 alone (-KChIP2.1) and with KChIP2.1 added via the bicistronic vector in HEK293 cells. A. Exemplar whole-cell currents elicited by 500 ms-depolarization pulses from −60 mV to +40 mV in 10 mV increments from a holding potential of −80 mV. B. The peak current at all potentials was increased by bicistronic addition of KChIP2.1 and the current activated at ~ − 30 mV as expected for Kv4.3 in the presence of KChIP2.1. C. The fast time constant of I to,f inactivation as a function of test potential (Kv4.3 alone: 7.66 ± 1.5 ms, n = 5 vs. Bicistronic Kv4.3/KChIP2.1: 21.2 ± 1.6 ms, n = 18 at +40 mV; p

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

    doi: 10.1016/j.yjmcc.2021.11.004

    Figure Lengend Snippet: Biophysical properties of I to,f currents produced by Kv4.3 alone (-KChIP2.1) and with KChIP2.1 added via the bicistronic vector in HEK293 cells. A. Exemplar whole-cell currents elicited by 500 ms-depolarization pulses from −60 mV to +40 mV in 10 mV increments from a holding potential of −80 mV. B. The peak current at all potentials was increased by bicistronic addition of KChIP2.1 and the current activated at ~ − 30 mV as expected for Kv4.3 in the presence of KChIP2.1. C. The fast time constant of I to,f inactivation as a function of test potential (Kv4.3 alone: 7.66 ± 1.5 ms, n = 5 vs. Bicistronic Kv4.3/KChIP2.1: 21.2 ± 1.6 ms, n = 18 at +40 mV; p

    Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

    Techniques: Produced, Plasmid Preparation

    Characterization of I to,f currents encoded by the bicistronic Kv4.3/KChIP2.1construct in rabbit ventricular cardiomycytes. A. Typical patch-clamp recordings of I to currents from freshly isolated cardiomyocytes and 40 h after transduction. Currents were evoked by step depolarizations to potentials between −30 and + 40 mV (in 10 mV increments). B. The left panel shows current-voltage relationships of intrinsic I to (n/ N = 7/3) and exogenous I to,f (n/ N = 16/8) and the fast time constant of I to,f inactivation (right hand panel). C. Concentration-dependence of the I to,f block by 4-AP block (n/N = 7/4). Exemplar recordings of the I to,f currents at +30 mV before and after application of 1 mmol/l of 4-AP are shown in the inset. D. Exemplar I to,f traces recorded from a transduced myocyte under basal conditions and in the presence of NS5806 (5 μmol/l). The right hand panel shows the augmentation of the mean peak I to,f current-voltage relationship by 5 μmol/l NS5806 ( p

    Journal: Journal of Molecular and Cellular Cardiology

    Article Title: Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

    doi: 10.1016/j.yjmcc.2021.11.004

    Figure Lengend Snippet: Characterization of I to,f currents encoded by the bicistronic Kv4.3/KChIP2.1construct in rabbit ventricular cardiomycytes. A. Typical patch-clamp recordings of I to currents from freshly isolated cardiomyocytes and 40 h after transduction. Currents were evoked by step depolarizations to potentials between −30 and + 40 mV (in 10 mV increments). B. The left panel shows current-voltage relationships of intrinsic I to (n/ N = 7/3) and exogenous I to,f (n/ N = 16/8) and the fast time constant of I to,f inactivation (right hand panel). C. Concentration-dependence of the I to,f block by 4-AP block (n/N = 7/4). Exemplar recordings of the I to,f currents at +30 mV before and after application of 1 mmol/l of 4-AP are shown in the inset. D. Exemplar I to,f traces recorded from a transduced myocyte under basal conditions and in the presence of NS5806 (5 μmol/l). The right hand panel shows the augmentation of the mean peak I to,f current-voltage relationship by 5 μmol/l NS5806 ( p

    Article Snippet: Specificity controls for KChIP2 labelling in transduced myocytes were performed by pre-incubating the anti-KChIP2 antibody with an immunizing peptide (Alomone) comprised of the amino acid sequences corresponding to the KChIP2 epitope at a 1:10 ratio (wt:wt) overnight at 4 °C (see Fig. S2A,B).

    Techniques: Patch Clamp, Isolation, Transduction, Concentration Assay, Blocking Assay