Journal: The FASEB Journal

Article Title: Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex

doi: 10.1096/fj.201902010RR

Figure Lengend Snippet: Effects of NS5806 on cloned Kv4.3/KChIP2/DPP6‐L channels in HEK293 cells. A, Representative current traces elicited by the depolarizing voltage steps from −40 to +40 mV for 2 seconds from a holding potential of −80 mV at different transfection ratios of Kv4.3: KChIP2: DPP6‐L (Left). The superimposed current traces at +40 mV in the absence and presence of NS5806 are shown on the right. B, Effect of 10 μM NS5806 on the peak current of the Kv4.3/KChIP2/DPP6‐L currents produced by different subunit transfection ratios, measured at +40 mV. C, The time constant of inactivation ( τ ) of Kv4.3/KChIP2/DPP6‐L currents produced by different subunit transfection ratios (n = 22). D, I–V relationships of Kv4.3/KChIP2/DPP6‐L peak current density at plasmid ratio 1:1:1 before and after 10 μM NS5806 (n = 18, * P < .05, ** P < .01 vs control). E, Steady‐state inactivation curves for Kv4.3/KChIP2/DPP6‐L channel complex at plasmid ratio 1:1:1 before and after 10 μM NS5806 (n = 18). F, Recovery from inactivation curves for Kv4.3/KChIP2/DPP6‐L channel complex at plasmid ratio1:1:1 before and after 10 μM NS5806 (n = 18)

Article Snippet: The following primary antibodies were used: anti‐DPP6 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 400‐550 of human DPP6, Abcam, UK); anti‐Kv1.4 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 589‐655 of rat Kv1.4 located in intracellular C‐terminus); anti‐Kv4.3 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 451‐468 of human Kv4.3 located in intracellular C‐terminus); anti‐KChIP2 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 2‐15 of human KChIP2 located in intracellular N‐terminus) (Alomone Labs, Israel); and anti‐GAPDH (Proteintech, Wuhan, China).

Techniques: Clone Assay, Transfection, Produced, Plasmid Preparation

Journal: The FASEB Journal

Article Title: Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex

doi: 10.1096/fj.201902010RR

Figure Lengend Snippet: Analysis on the putative interactions between KChIP2 and DPP6‐L. A 1 , Modeling and docking simulation of putative interactions between DPP6‐Lin and KChIP2 . Homology model of KChIP2. A 2 , The top‐ranked model of the intracellular domain of DPP6‐L (DPP6‐Lin). A 3 , Top‐ranked models of both proteins. A 4 , Best scored model of docking KChIP2 with DPP6‐Lin; putative‐interacting residues are indicated. B 1 , Schematic depiction of DPP6‐L and the location of mutated residues within the putative KChIP2 interaction site. B 2 , Representative recordings of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut currents from HEK293 cells using 500 ms square voltage pulses (from −40 to +40 mV; holding potential is −80 mV). B 3 , The time constants of inactivation ( τ ) of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut current traces plotted against voltage (** P < .01). C 1 , Representative recordings of I Kv4.3/KChIP2/DPP6‐L‐WT and I Kv4.3/KChIP2/DPP6‐L‐Mut in HEK293 cells before and after 10 μM NS5806. C 2 , Summary data for the effect of NS5806 on the current amplitudes of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut channels (* P < .05). C 3 , Summary data for the effect of NS5806 on the current inactivation kinetics (at +40 mV) of Kv4.3/KChIP2/DPP6‐L‐WT and Kv4.3/KChIP2/DPP6‐L‐Mut channels (** P < .01)

Article Snippet: The following primary antibodies were used: anti‐DPP6 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 400‐550 of human DPP6, Abcam, UK); anti‐Kv1.4 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 589‐655 of rat Kv1.4 located in intracellular C‐terminus); anti‐Kv4.3 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 451‐468 of human Kv4.3 located in intracellular C‐terminus); anti‐KChIP2 (a polyclonal antibody raised in rabbit against a purified peptide corresponding to amino acid residues 2‐15 of human KChIP2 located in intracellular N‐terminus) (Alomone Labs, Israel); and anti‐GAPDH (Proteintech, Wuhan, China).

Techniques:

Journal:

Article Title: PPAR?-Mediated Remodeling of Repolarizing Voltage-Gated K + (Kv) Channels in a Mouse Model of Metabolic Cardiomyopathy

doi: 10.1016/j.yjmcc.2008.03.023

Figure Lengend Snippet: (A–G) Representative Western blots of fractionated (5–6 week old) adult WT (n = 3) and MHC-PPARα (n = 4) mouse ventricular proteins probed with a polyclonal anti-Kv4.2 (A), a monoclonal anti-KChIP2 (B), polyclonal anti-TASK1 (C), anti-TASK2 (D), anti-Cx43 (E), anti-Cx45 (F) antibodies or with a monoclonal anti-transferrin receptor (G) antibody. The arrows denote the protein bands corresponding to the proteins targeted by the antibodies, and the bands that were scanned for quantification of protein expression levels. (H) Films from Western blots completed on samples from (5–6 week) MHC-PPARα (n = 10) and WT (n = 10) ventricles were scanned, and the densities of the Kv4.2, KChIP2, TASK1, TASK2, Cx43, and Cx45, as well as of the transferrin receptor, bands were measured. The densities of the bands in the MHC-PPARα samples were normalized to the densities of the corresponding bands in the WT samples. As is evident, Kv4.2 and KChIP2 expression were significantly (* P < 0.01) reduced in MHC-PPARα, compared to WT, ventricles, whereas there are no significant differences in TASK1 or TASK2 expression in MHC-PPARα and WT ventricles. In addition, Cx43 protein, but not Cx45 protein, expression is markedly reduced in MHC-PPARα, compared with WT, ventricles.

Article Snippet: The polyclonal rabbit anti-Kv4.2 antibody (Chemicon, Temecula, CA) and the monoclonal anti-KChIP2 antibody (UC Davis NINDS/NIMH Neuromab Facility, Davis, CA) have been tested previously for specificity and cross reactivity [ 26 , 27 ].

Techniques: Western Blot, Expressing

Journal:

Article Title: PPAR?-Mediated Remodeling of Repolarizing Voltage-Gated K + (Kv) Channels in a Mouse Model of Metabolic Cardiomyopathy

doi: 10.1016/j.yjmcc.2008.03.023

Figure Lengend Snippet: RNA Preparation and SYBR Green Quantitative RT-PCR

Article Snippet: The polyclonal rabbit anti-Kv4.2 antibody (Chemicon, Temecula, CA) and the monoclonal anti-KChIP2 antibody (UC Davis NINDS/NIMH Neuromab Facility, Davis, CA) have been tested previously for specificity and cross reactivity [ 26 , 27 ].

Techniques: SYBR Green Assay, Quantitative RT-PCR

Journal: JCI Insight

Article Title: Indoxyl sulfate reduces I to,f by activating ROS/MAPK and NF- κ B signaling pathways

doi: 10.1172/jci.insight.145475

Figure Lengend Snippet: ( A ) Flow chart of animal experiments. ( B and D ) Determinations of IS levels in serum ( B ) and heart tissues ( D ) at 8 weeks after right nephrectomy ( n = 5 per group). ( C and E ) Measurements of IS levels in serum ( C ) and heart tissues ( E ) after 8 weeks of IS treatment ( n = 6 per group). ( F and I ) Representative immunoblots ( F ) and average data ( I ) of Kv4.2, Kv4.3, and KChIP2 proteins in vehicle and IS treatment groups ( n = 6 per group). ( G and H ) Representative immunoblots ( G ) and average data ( H ) of Kv4.2, Kv4.3, and KChIP2 proteins in sham, CKD, and CKD plus BB536 groups ( n = 5 per group). ( J ) Representative IHC images of Kv4.2 and Kv4.3 proteins in rats. Scale bar: 100 μm. Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test ( C , E , and I ) and 1-way ANOVA followed by Bonferroni post hoc test ( B , D , and H ). * P < 0.05, ** P < 0.01.

Article Snippet: The cells were then incubated with primary antibodies including anti-cTNI antibody (1:100, 21652-1-AP, Proteintech), anti-Kv4.2 antibody (1:100, 21298-1-AP, Proteintech), anti-KChIP2 antibody (1:100, ab88542, Abcam), and anti–NF-κB p65 antibody (1:400, 8242S, Cell Signaling Technology) at 4°C overnight.

Techniques: Western Blot

Journal: JCI Insight

Article Title: Indoxyl sulfate reduces I to,f by activating ROS/MAPK and NF- κ B signaling pathways

doi: 10.1172/jci.insight.145475

Figure Lengend Snippet: ( A and B ) Representative immunoblots ( A ) and average data ( B ) of Kv4.2, Kv4.3, and KChIP2 proteins in NRVMs treated with different concentrations of IS ( n =3 per group). ( C ) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in NRVMs treated with different concentrations of IS ( n = 3 per group). ( D and E ) Representative immunofluorescence images of Kv4.2 ( D ) and KChIP2 ( E ) proteins in NRVMs. Scale bar: 50 μm. ( F and G ) Representative I to,f traces ( F ) and average I to,f densities (peak minus steady state) versus membrane potentials ( G ) in NRVMs treated with different concentrations of IS ( n = 5 per group).The inset in F shows the voltage-clamp protocol. ( H ) Average time constants (τ) of decay of I to,f at +60 mV in NRVMs ( n = 5 per group). ( I and J ) Average values of constants (k) of activation ( I ) and half-maximal voltage of activation(V 0.5 , act) ( J ) in NRVMs ( n = 5 per group). ( K ) Voltage-dependent activation curves of I to,f in NRVMs ( n = 5 per group). Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Bonferroni post hoc test. * P < 0.05 versus control, ** P < 0.01 versus control.

Article Snippet: The cells were then incubated with primary antibodies including anti-cTNI antibody (1:100, 21652-1-AP, Proteintech), anti-Kv4.2 antibody (1:100, 21298-1-AP, Proteintech), anti-KChIP2 antibody (1:100, ab88542, Abcam), and anti–NF-κB p65 antibody (1:400, 8242S, Cell Signaling Technology) at 4°C overnight.

Techniques: Western Blot, Immunofluorescence, Activation Assay

Journal: JCI Insight

Article Title: Indoxyl sulfate reduces I to,f by activating ROS/MAPK and NF- κ B signaling pathways

doi: 10.1172/jci.insight.145475

Figure Lengend Snippet: ( A and D ) Measurements of ROS productions based on DHE fluorescence in rat hearts. ( A ) Representative images of DHE immunofluorescence. Scale bar: 200 μm. ( D ) Relative ROS fluorescence intensities in sham, CKD, and CKD plus BB536 groups ( n = 5 per group), and in vehicle and IS treatment groups ( n = 6 per group). ( B and E ) Measurements of ROS productions based on flow cytometry in NRVMs treated with different concentrations of IS. ( B ) Representative flow cytometric histograms in NRVMs. ( E ) Relative ROS fluorescence intensities in NRVMs ( n = 5 per group). ( C and F ) NAC reversed ROS production induced by IS in NRVMs. ( C ) Representative flow cytometric histograms in 4 groups. ( F ) Relative ROS fluorescence intensities detected by flow cytometry in 4 groups ( n = 5 per group). ( G and H ) Representative immunoblots of NOX2 proteins in sham, CKD, and CKD plus BB536 groups ( n = 5 per group) ( G ), and in vehicle and IS treatment groups ( n = 6 per group) ( H ). ( I ) Average immunoblots data of NOX2 proteins in the hearts of rats. ( J and M ) Representative immunoblots ( J ) and average data ( M ) of NOX2 proteins in NRVMs treated with different concentrations of IS ( n = 3 per group). ( K and N ) Representative immunoblots ( K ) and average data ( N ) of NOX2 proteins in control, DPI, IS, and IS plus DPI groups ( n = 3 per group). ( L and O ) Representative immunoblots ( L ) and average data ( O ) of NOX2 proteins in control, APO, IS, and IS plus APO groups ( n = 3 per group). ( P and Q ) Representative immunoblots ( P ) and average data ( Q ) of Kv4.2, Kv4.3, and KChIP2 proteins in control, NAC, IS, and IS plus NAC groups ( n = 3 per group). ( R ) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, NAC, IS, and IS plus NAC groups ( n = 3 per group). NRVMs in IS and IS plus NAC groups were treated with 10 μM IS. Data are presented as mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test ( D and I ) and 1-way ANOVA followed by Bonferroni post hoc test ( D – F , I , M – O , Q , and R ). * P < 0.05, ** P < 0.01.

Article Snippet: The cells were then incubated with primary antibodies including anti-cTNI antibody (1:100, 21652-1-AP, Proteintech), anti-Kv4.2 antibody (1:100, 21298-1-AP, Proteintech), anti-KChIP2 antibody (1:100, ab88542, Abcam), and anti–NF-κB p65 antibody (1:400, 8242S, Cell Signaling Technology) at 4°C overnight.

Techniques: Fluorescence, Immunofluorescence, Flow Cytometry, Western Blot

Journal: JCI Insight

Article Title: Indoxyl sulfate reduces I to,f by activating ROS/MAPK and NF- κ B signaling pathways

doi: 10.1172/jci.insight.145475

Figure Lengend Snippet: ( A and B ) Representative immunoblots ( A ) and average data ( B ) of Kv4.2, Kv4.3, KChIP2, and P-p38 MAPK in control, SB, IS, and IS plus SB groups ( n = 3 per group). ( C ) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, SB, IS, and IS plus SB groups ( n = 3 per group). ( D and E ) Representative immunoblots ( D ) and average data ( E ) of Kv4.2, Kv4.3, KChIP2, and P-p44/42 MAPK in control, U0126, IS, and IS plus U0126 groups ( n = 3 per group). ( F ) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, U0126, IS, and IS plus U0126 groups ( n = 3 per group). ( G and H ) Representative immunoblots ( G ) and average data ( H ) of Kv4.2, Kv4.3, KChIP2, and P-NF-κB in control, BAY, IS, and IS plus BAY groups ( n = 3 per group). ( I ) Relative mRNA expressions for Kv4.2, Kv4.3, and KChIP2 in control, BAY, IS, and IS plus BAY groups ( n = 3 per group). NRVMs in IS, IS plus SB, IS plus U0126, and IS plus BAY groups were treated with 10 μM IS. Data are presented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA, followed by Bonferroni post hoc test. * P < 0.05, ** P < 0.01.

Article Snippet: The cells were then incubated with primary antibodies including anti-cTNI antibody (1:100, 21652-1-AP, Proteintech), anti-Kv4.2 antibody (1:100, 21298-1-AP, Proteintech), anti-KChIP2 antibody (1:100, ab88542, Abcam), and anti–NF-κB p65 antibody (1:400, 8242S, Cell Signaling Technology) at 4°C overnight.

Techniques: Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Notch signaling modulates the electrical behavior of cardiomyocytes

doi: 10.1152/ajpheart.00587.2016

Figure Lengend Snippet: Primers for PCR analysis

Article Snippet: Thin sections of the left ventricle (LV) were indirectly immunolabeled for the detection of GFP (Anti-GFP Antibody, catalog no. ab5450, Abcam) and KChIP2 (KCNIP2 Polyclonal Antibody, catalog no. PA5-41075, ThermoFisher Scientific).

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Notch signaling modulates the electrical behavior of cardiomyocytes

doi: 10.1152/ajpheart.00587.2016

Figure Lengend Snippet: Notch signaling activation reduces Kv channel-interacting protein 2 (KChIP2) expression in cardiomyocytes. A: KChIP2 (white) expression in the left ventricular (LV) myocardium of a MCM-WT mouse. Nuclei are stained by DAPI. Autofluorescence (AF) in the green channel was collected to visualize the structure of myocardial tissue. The green channel is omitted at right. B: green fluorescent protein (GFP; green) and KChIP2 (white) expression in the LV myocardium of a MCM-NICD-GFP mouse. Nuclei are stained by DAPI. The green channel is omitted at right. C: quantification of KChIP2 levels in myocytes in LV tissue of MCM-WT mice [wild type (WT), n = 2 mice] and MCM-NICD-GFP mice [Notch1 intracellular domain (NICD), n = 3 mice]. For each acquired image, the intensity of the fluorescent signal (F) from individual myocytes was normalized with respect to the signal of the myocyte presenting the highest fluorescent intensity (Fmax). Normalized data are presented as F/Fmax. *P < 0.001 vs. WT. D: quantification of relative KChIP2 levels in NICD-GFP-negative and NICD-GFP-positive myocytes in the myocardium of the MCM-NICD-GFP mice (NICD) shown in C. *P < 0.001 vs. NICD-GFP negative.

Article Snippet: Thin sections of the left ventricle (LV) were indirectly immunolabeled for the detection of GFP (Anti-GFP Antibody, catalog no. ab5450, Abcam) and KChIP2 (KCNIP2 Polyclonal Antibody, catalog no. PA5-41075, ThermoFisher Scientific).

Techniques: Activation Assay, Expressing, Staining