Journal: eLife

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

doi: 10.7554/eLife.80710

Figure Lengend Snippet: ( A and B ) Relative protein expression of Kv4.3 and KChiP2 subunits determined by western blotting in left ventricular samples of SED (n=12) and TRN (n=12) dogs, respectively. ( C ) Representative image of Kv4.3 and KChIP2 bands and their corresponding loading controls (GAPDH). ( D and E ) Relative densities of Kv4.3 and KChiP2 protein immunolabeling obtained from SED (n=30 cells/6 dogs) and TRN (n=30 cells/6 dogs) cardiomyocytes. ( F ) Representative immunofluorescence images of canine cardiomyocytes with Kv4.3 and KChiP2 immunolabeling. The ‘n’ numbers refer to the number of dogs ( A and B ) or the number of cells followed by the number of dogs from which the cells were obtained ( D and E ). Data are expressed as mean ± SEM. Blue dots represent individual data. Figure 6—source data 1. Relative protein expression of Kv4.3 subunit determined by western blotting in sedentary and trained dogs. Figure 6—source data 2. Relative protein expression of KChiP2 subunit determined by western blotting in sedentary and trained dogs. Figure 6—source data 3. Relative density of Kv4.3 subunit determined by immunocytochemistry in sedentary and trained dogs. Figure 6—source data 4. Relative density of KChiP2 subunit determined by immunocytochemistry in sedentary and trained dogs. Figure 6—source data 5. Original unedited membranes of western blots with the relevant bands clearly labeled. Figure 6—source data 6. Original files of the full raw unedited membranes of western blots.

Article Snippet: The membrane was blocked with 2.5% non-fat milk for 1 hr at room temperature and immunolabeled overnight at 4°C with anti-KChIP2 (Alomone, #APC-142, RRID: AB_2756744 ) and anti-Kv4.3 (Alomone, #APC-017, RRID: AB_2040178 ) primary antibodies diluted 1:1000.

Techniques: Expressing, Western Blot, Immunolabeling, Immunofluorescence, Immunocytochemistry, Labeling

Journal: Journal of Biological Chemistry

Article Title: A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels

doi: 10.1074/jbc.ra118.006549

Figure Lengend Snippet: Figure 5. Ca2+ regulation of Kv4.2-KChIP2 complexes is KChIP isoform-dependent. (a) Consensus protein domain organization of KChIP family members (top). Protein domain organization of KChIP2 isoforms that demonstrates N-terminal variability (below). (b) Two-way ANOVA returned significant differences in peak Kv4.2 current density between KChIP2 isoforms (p = 0.0005), by treatment (p = 0.0227), and the interaction (p = 0.0150). However, Kv4.2 peak current density was unaffected by intracellular Ca2+ when expressed with long forms of KChIP2 (KChIP2a1: p = 0.9415 KChIP2a: p = 0.9997), whereas Kv4.2 peak current was significantly increased in the presence of Ca2+ for shorter forms of KChIP2 (KChIP2b: p = 0.0114; KChIP2c: p = 0.0203). (c) Sequence alignment of human N-terminal domains of KChIP2 isoforms. The putative polybasic domain conserved in Ca2+-insensitive KChIP isoforms is underlined and basic residues are indicated with an asterisk. (d) Site-directed acidification of the putative polybasic motif in KChIP2a1 rescues Ca2+ enhancement of peak current density. Two-way ANOVA returned no differences between WT KChIP2a1 and mutant KChIP2a1 groups by construct expression ( p = 0.1419) or treatment (p = 0.6426), however, the interaction was significant (p = 0.0124) likely due to differences in peak current density between WT and mutant KChIP2a1. Sidak’s multiple comparison revealed that mutant KChIP2a1 responded to Ca2+ (p = 0.0107) while WT KChIP2a1 did not (p = 0.6970) as also shown in (b). Error bars represent mean +/- SEM. * = p<0.05, *** = p<0.001 by two-way ANOVA and Sidak’s multiple comparison test. Refer to Table 2 for numerical data and replicate information. 24

Article Snippet: Kv4 auxiliary subunit expression was carried out using Human DPP6 (RC216919), Human KChIP1a (RC224442), 1b (RC208255), 2a1 (RC213131), 2b (RC203823), 3a (RC203957), 4bL (RC211488), 4a (RC211613), and Mouse KChIP2a (MC211934) were obtained by guest on January 8, 2019 http://w w w .jbc.org/ D ow nloaded from Ca2+ regulation of KChIP-Kv4 channel complexes from Origene.

Techniques: Sequencing, Mutagenesis, Construct, Expressing, Comparison

Journal: Cardiovascular research

Article Title: The cAMP response element binding protein modulates expression of the transient outward current: implications for cardiac memory.

doi: 10.1016/j.cardiores.2005.05.028

Figure Lengend Snippet: Fig. 3. Panel A: Representative Western blots of three memory dogs and three sham dogs. Loading was controlled for Histone 1. Panel B: Western blots illustrating the difference between epicardial and endocardial KChIP2 levels and the reduced epi- to endocardial gradient in CM. Loading was controlled for GAPDH. M=memory; S=sham.

Article Snippet: The following primary antibodies and their appropriate secondary antibodies were used: a monoclonal KChIP2 antibody (a gift of Dr. Mark Bowlby), CREB (cell signaling), CREM (Santa Cruz, Santa Cruz, CA), GAPDH (Research diagnostics, Flanders, NJ), histone 1 (Santa Cruz), AT1 (Santa Cruz).

Techniques: Western Blot

Journal: Cardiovascular research

Article Title: The cAMP response element binding protein modulates expression of the transient outward current: implications for cardiac memory.

doi: 10.1016/j.cardiores.2005.05.028

Figure Lengend Snippet: Fig. 4. Electrophoretic mobility shift assay (EMSA) demonstrating the binding of CREB to the promoter region of KChIP2: Panel A: EMSA with HeLa cell nuclear extract binding to the KChIP2 CRE (K2CRE). Nuclear protein binds to K2CRE and forms a specific probe-protein complex (arrow). This binding is competed by 100 fold molar excess of the same, but unlabeled, probe (K2CRE) or an unlabeled consensus CRE (CRE). This complex is recognized by an antiserum against pCREB which competes with the probe for binding CREB (demonstrated by loss of band density and appearance of unbound probe in the lane). An unrelated antiserum (ERG antibody) does not recognize the complex, showing the pCREB specificity of the competition. The right lane shows a control experiment without nuclear extract. Panel B: EMSA of Hela cell nuclear extract binding to the consensus CRE and competed by the same consensus CRE or K2CRE. The specific CREB containing nuclear protein-DNA complex (arrow) is competed similarly by the CRE and KCRE probes indicating that K2CRE and CRE bind CREB with comparable affinity. Protein DNA complexes were competed at 5, 20 and 100 molar excesses, respectively. Experiments were performed three independent times with identical results.

Article Snippet: The following primary antibodies and their appropriate secondary antibodies were used: a monoclonal KChIP2 antibody (a gift of Dr. Mark Bowlby), CREB (cell signaling), CREM (Santa Cruz, Santa Cruz, CA), GAPDH (Research diagnostics, Flanders, NJ), histone 1 (Santa Cruz), AT1 (Santa Cruz).

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca 2+ regulation of Kv4 channels

doi: 10.1074/jbc.RA118.006549

Figure Lengend Snippet: Ca2+ regulation of Kv4.2–KChIP2 complexes is KChIP isoform-dependent. a, consensus protein domain organization of KChIP family members (top). Protein domain organization of four KChIP2 isoforms that demonstrates N-terminal variability (below). b, two-way ANOVA returned significant differences in peak Kv4.2 current density between KChIP2 isoforms (p = 0.0005), by treatment (p = 0.0227), and the interaction (p = 0.0150). However, Kv4.2 peak current density was unaffected by intracellular Ca2+ when expressed with long forms of KChIP2 (KChIP2a1, p = 0.9415; KChIP2a, p = 0.9997), whereas Kv4.2 peak current was significantly increased in the presence of Ca2+ for shorter forms of KChIP2 (KChIP2b, p = 0.0114; KChIP2c, p = 0.0203). c, sequence alignment of human N-terminal domains of KChIP2 isoforms. The putative polybasic domain conserved in Ca2+-insensitive KChIP isoforms is underlined, and basic residues are indicated with an asterisk. d, site-directed acidification of the putative polybasic motif in KChIP2a1 rescues Ca2+ enhancement of peak current density. Two-way ANOVA returned no differences between WT KChIP2a1 and mutant KChIP2a1 groups by construct expression (p = 0.1419) or treatment (p = 0.6426); however, the interaction was significant (p = 0.0124) likely due to differences in peak current density between WT and mutant KChIP2a1. Sidak's multiple comparison revealed that mutant KChIP2a1 responded to Ca2+ (p = 0.0107), whereas WT KChIP2a1 did not (p = 0.6970) as also shown in b. Error bars, mean ± S.E. *, p < 0.05; ***, p < 0.001 by two-way ANOVA and Sidak's multiple comparison test. Refer to Table 2 for numerical data and replicate information.

Article Snippet: Kv4 auxiliary subunit expression was carried out using human DPP6 (RC216919), human KChIP1a (RC224442), KChIP1b (RC208255), KChIP2a1 (RC213131), KChIP2b (RC203823), KChIP3a (RC203957), KChIP4bL (RC211488), KChIP4a (RC211613), and mouse KChIP2a (MC211934) (Origene).

Techniques: Sequencing, Mutagenesis, Construct, Expressing, Comparison

Journal: Oncotarget

Article Title: A novel KCND3 mutation associated with early-onset lone atrial fibrillation

doi: 10.18632/oncotarget.23303

Figure Lengend Snippet: (A) Typical currents recorded from HEK293 cells transfected WT or T361S of K v 4.3 together with KChIP2 in response to increasing step potential. (B) Current-voltage relationship. The protocol was shown in the inset. Data was shown as means ± SEM.

Article Snippet: T vectors containing cDNA encoding human KChIP2 (NM_173192.2) or human KCND3 (NM_172198.2) were purchased from Proteintech Company (Wuhan, China).

Techniques: Transfection

Journal: Oncotarget

Article Title: A novel KCND3 mutation associated with early-onset lone atrial fibrillation

doi: 10.18632/oncotarget.23303

Figure Lengend Snippet: (A) Cell surface biotinylation assays were performed in Hek293 cells co-transfected KChIP2 with wild type or mutant T361S of K v 4.3 after 48 hours. Intergrin α5 was used as loading to calibrate the cell-surface proteins. No GAPDH was detected in the biotinylated fraction (data not shown). (B) The total expression of K v 4.3 protein was extracted from HEK293 cells co-transfected KChIP2 with wild type or mutant T361S of K v 4.3. This experiment was repeated at least three times, and similar results were obtained. Data was shown as means ± SEM. * P < 0.05.

Article Snippet: T vectors containing cDNA encoding human KChIP2 (NM_173192.2) or human KCND3 (NM_172198.2) were purchased from Proteintech Company (Wuhan, China).

Techniques: Transfection, Mutagenesis, Expressing

Journal: Acta pharmacologica Sinica

Article Title: Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

doi: 10.1038/s41401-021-00659-8

Figure Lengend Snippet: Fig. 4 Effects of IL-17 knockout on the Ito and KChIP2 expression in the hearts of diabetic mice. a Representative traces of the Ito. b Current density-voltage (I–V) relationship of the Ito. n = 8–11 cells. c Current density-voltage (I–V) relationship of the Iss. n = 9–16 cells. d Mean membrane capacitance of the Ito and Iss n = 9–16 cells. e The protein and mRNA levels of Kv4.2. n = 7. f The protein and mRNA levels of Kv4.3. n = 7. g The protein and mRNA levels of KChIP2. n = 5. *P < 0.05 vs. WT mice; #P < 0.05 vs. WT+DM mice. WT wild-type, IL-17 KO IL-17 knockout, WT+DM wild-type+diabetes mellitus, IL-17 KO+DM IL-17 knockout+diabetes mellitus.

Article Snippet: The primary antibodies included rabbit anti-mouse Nav1.5 (Alomone Labs, Israel), Kv4.2 (Alomone Labs, Israel), Kv4.3 (Alomone Labs, Israel), KChIP2 (Boster, China), Cav1.2 (Alomone Labs, Israel), and NF-κB (CST, USA). β-Actin was used as an internal control.

Techniques: Knock-Out, Expressing, Membrane

Journal: Acta pharmacologica Sinica

Article Title: Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

doi: 10.1038/s41401-021-00659-8

Figure Lengend Snippet: Fig. 8 Knockout of IL-17 protects against ventricular arrhythmias in STZ-induced diabetic mice. Knockout of IL-17 downregulates the expression of NF-κB, which suppresses the expression of KCNIP2, which encodes potassium voltage-gated channel interacting protein 2; CACNA1C, which encodes the pore-forming subunit of the voltage-gated L-type calcium channel Cav1.2; and SCN5A, which encodes the pore-forming subunit of the voltage-gated sodium channel Nav1.5. Decreased expression of KChIP2 and Nav1.5 prolonged the APD and slowed the conduction velocity, which increased susceptibility to ventricular arrhythmias. KCNIP2, potassium voltage-gated channel interacting protein 2; CACNA1C, calcium voltage-gated channel subunit alpha 1 C; KCND2, potassium voltage-gated channel subfamily D member 2; KCND3, potassium voltage-gated channel subfamily D member 3; SCN5A, sodium voltage-gated channel alpha subunit 5; WT+DM, wild-type+diabetes mellitus; IL-17 KO+DM, IL-17 knockout+diabetes mellitus.

Article Snippet: The primary antibodies included rabbit anti-mouse Nav1.5 (Alomone Labs, Israel), Kv4.2 (Alomone Labs, Israel), Kv4.3 (Alomone Labs, Israel), KChIP2 (Boster, China), Cav1.2 (Alomone Labs, Israel), and NF-κB (CST, USA). β-Actin was used as an internal control.

Techniques: Knock-Out, Expressing