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: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: To examine the expression of KChIP transcripts, RNA was extracted from samples prepared from the posterior (~1 mm) cortex (containing visual cortex) of adult WT mice. (A) Using sequence specific primer pairs and RT-PCR, transcripts coding for KChIP2, KChIP3, and KChIP4 were readily detected. Experiments were also performed to examine KChIP protein expression and association with Kv4.2. Immunoprecipitations using a rabbit anti-Kv4.2 antibody were conducted on lysates prepared from the posterior cortices of WT or Kv4.2−/− mice and blots of fractionated immunoprecipitated samples were probed using subunit specific antibodies. (B) In immunoprecipitated samples from WT cortices, but not in those from Kv4.2−/− cortices, both Kv4.2 and Kv4.3 were readily detected. (C) In immunoprecipitated samples from WT, but not Kv4.2−/−, cortices, KChIP2, KChIP3 and KChIP4 were also detected indicating that all three KChIP proteins are expressed and assemble with Kv4.2 in cortex. Molecular masses are indicated on the blots in kDa.

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal:

Article Title: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: (A) Lysates prepared from the posterior (~1 mm) cortices of WT, KChIP2−/− and KChIP3−/− mice (n=6 animals for each genotype) were fractionated, transferred to PVDF membranes and probed with a specific anti-KChIP2, anti-KChIP3 or anti-KChIP4 antibody. All three KChIPs were detected in samples from WT mice. Confirming the specificities of the anti-KChIP2 and anti-KChIP3 antibodies, no signal was detected with the anti-KChIP2 or the anti-KChIP3 antibody in samples from KChIP2−/− or KChIP 3−/− cortices, respectively. Blots were also probed with antibodies against β-tubulin to confirm equal loading of proteins. In each lane, anti-KChIP antibody signals were quantified and normalized to the anti-β-tubulin antibody signals. (B) In KChIP2−/− cortices, the mean ± SEM expression levels of KChIP3 and KChIP4 proteins were significantly (+ p<0.01) higher than in WT cortices. Similarly, the mean ± SEM expression levels of the KChIP2 and KChIP4 proteins were significantly (*p<0.05 and +p<0.01, respectively) higher in KChIP3−/− cortices. (C) QRT-PCR analysis revealed that the mean ± SEM expression level of KChIP2 transcript was not significantly different in WT (n=6) and KChIP3−/− (n=6) cortices, whereas the mean ± SEM expression level of KChIP4 transcript was slightly, but significantly (*p>0.05), higher in cortices from KChIP3−/−, compared with WT, mice. Molecular masses are indicated on the blots in kDa.

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR

Journal:

Article Title: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: Lysates were prepared from the posterior (~1 mm) cortices of WT, KChIP2−/− and KChIP3−/− mice (n=6 of each genotype) and fractionated by SDS-PAGE. Following transfer, membranes were probed with a monoclonal anti-Kv4.2 or anit-Kv4.3 antibody and, subsequently, with an anti-GAPDH antibody, to verify equal loading of proteins in each lane. Signals from the anti-Kv4.2 and anti-Kv4.3 antibodies in each lane were quantified and normalized to signals from the anti-GAPDH antibody in the same lane. Molecular masses are indicated on the blots in kDa. (B) Mean ± SEM levels of Kv4.2 and Kv4.3 proteins are not significantly different in either KChIP2−/− or KChIP3−/−, compared to WT, cortices.

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: SDS Page

Journal:

Article Title: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: As described in Materials and Methods, plasmids encoding human miR30, with substituted targeting sequences and a fluorescent protein (YFP, CFP or tdTomato) on a single transcript, were generated. (A) The miR30 sequence was placed on an intron downstream of the CMV promoter and upstream of the sequence coding for the fluorescent protein (CFP, YFP or tdTomato). (B) Transfections of these plasmids into neurons allowed for visual identification of neurons expressing one or all three of the plasmids for subsequent electrophysiological recording. (C) Specific sequences targeting KChIP2, KChIP3 and KChIP4 were screened in HEK-293 cells. The targeted KChIP (KChIP2, 3 or 4) was co-expressed with either a control (non-targeting) miRNA construct or with a miRNA construct containing sequence complementary to the sequence of the targeted KChIP. Lysates were prepared from transfected HEK-293 cells, fractionated by SDS-PAGE, transferred to membranes and probed for KChIP2, KChIP3 or KChIP4. Blots were also probed with an anti-transferrin receptor (Transferrin R) antibody to verify equal loading of proteins. Targeting sequences found to reduce the expression of each of the targeted KChIPs are illustrated and were used in subsequent experiments, on cortical neurons.

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: Generated, Sequencing, Transfection, Expressing, Construct, SDS Page

Journal:

Article Title: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: To examine the combined role(s) of the KChIPs in the generation of Kv4-encoded IA channels, cortical pyramidal neurons were isolated from Kv1.4−/− mice and transfected with the validated miRNA constructs targeting KChIP2, KChIP3 and KChIP4 or with plasmids containing control (non-targeting) sequences. Because each KChIP miRNA construct also encoded for a distinct fluorescent protein (CFP, YFP or tdTomato) cells expressing all three KChIP targeting miRNA constructs could be identified. (A) Recordings were obtained from neurons expressing control plasmids or (B) all three targeting plasmids. Surprisingly, no prominent rapidly inactivating component was observed in about half (11 of 20) of the neurons expressing the KChIP targeting miRNA constructs and delayed rectifier currents were increased. In all cells, IA was isolated and quantified using the prepulse paradigm described in the legend to Figure 1. Analyses of subtracted records (a–b) revealed residual Kv4-encoded IA in all neurons expressing the three KChIP targeting miRNAs simultaneously. The mean ± SEM IA density was significantly (‡p<0.001) lower (C) in neurons expressing the three KChIP targeting miRNA constructs (n=20) than in neurons expressing control constructs (n=21). (D) Consistent with the upregulation of delayed rectifier currents, analysis of the peak current (IPeak) revealed no significant reduction in mean ± SEM IPeak density in neurons expressing KChIP targeting miRNA compared to those expressing control constructs, despite the marked reduction in IA densities (c).

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: Isolation, Transfection, Construct, Expressing

Journal:

Article Title: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: HEK-293 cells were transfected with DNA constructs encoding Kv4.2 alone (n=9), one of the KChIPs (KChIP2 n=6, KChIP3 n=9, KChIP4 n =6) alone, or Kv4.2 in combination with KChIP2 (n=6), KChIP3 (n=9) or KChIP4 (n=6). (A) Western Blots on lysates prepared from transfected HEK-293 cells were probed with the monoclonal anti-Kv4.2 antibody. Blots were also probed with anti-transferrin receptor antibody (Transferrin R) to verify equal loading of proteins in each lane. The anti-Kv4.2 antibody signals were measured and normalized to the signals from the anti-transferrin receptor in the same lane. (B) Quantitative analyses revealed a significant (*p<0.05) increase in Kv4.2 protein in cells expressing Kv4.2 plus one of the three KChIPs, compared to cells expressing Kv4.2 alone. (C) Western Blots conducted on HEK-293 cell lysates using the anti-KChIP2, anti-KChIP3 or anti-KChIP4 antibody also revealed that KChIP protein expression was increased in cells coexpressing Kv4.2, compared to cells express KChIP2, 3 or 4 alone. (D) Mean ± SEM levels of KChIP2, 3 and 4 protein expression were significantly (*p<0.05, +p<0.01) higher in cells coexpressing Kv4.2 compared to cells expressing either of the KChIP proteins alone.

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: Transfection, Construct, Western Blot, Expressing

Journal:

Article Title: Interdependent Roles for Accessory KChIP2, KChIP3 and KChIP4 Subunits in the Generation of Kv4-encoded I A Channels in Cortical Pyramidal Neurons

doi: 10.1523/JNEUROSCI.2487-10.2010

Figure Lengend Snippet: (A) Representative Western Blots of fractionated lysates prepared from posterior (~1 mm) cortices of WT (n=6), Kv4.2−/−(n=6), Kv4.3−/− (n=6) and Kv4.2−/−/Kv4.3−/− (n=3) mice were probed with the anti-KChIP antibodies. KChIP protein levels were differentially affected by the loss of Kv4.2 or Kv4.3, although drastic reductions in all three proteins were evident with the loss of both Kv4.2 and Kv4.3. For quantification, blots were also probed with an anti-β-tubulin antibody to confirm equal protein loading, in each lane, and signals from the anti-KChIP2, 3 or 4 antibodies were normalized against the signals from the anti-β tubulin antibody in the same lane. (B) Analysis of mean (± SEM) normalized data revealed that the expression levels of KChIP2, KChIP3 and KChIP4 proteins in Kv4.2−/− and Kv4.3−/− cortices were significantly (*p<0.05, +p<0.01 or ‡p<0.001) lower than in WT cortices. In Kv4.2−/−Kv4.3−/− cortices KChIP2, KChIP3 and KChIP4 protein expression levels were extremely low. (C) QRT-PCR of analysis of RNA isolated from the posterior cortices of WT (n=6), Kv4.2−/−(n=6), Kv4.3−/−(n=6) and Kv4.2−/−/Kv4.3−/− (n=3) mice revealed no reductions in KChIP transcripts. The mean ± SEM transcript expression level of KChIP4 was, however, significantly (p<0.05) higher in Kv4.3−/− and in Kv4.2−/−/Kv4.3−/−, compared with WT, cortices.

Article Snippet: The expression levels of KChIP2, 3 and 4 were determined using sequence specific primers (see below) and SYBR green (Applied Biosystems) for QRT-PCR; experiments were conducted on a 7900HT Fast Real Time PCR System (Applied Biosystems).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Isolation

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) Results of miRNA microarray showing the log 2 of the fold changes in miR expression following 72 hr of KChIP2 siRNA treatment. Arrow identifies miR-34b and −34c amongst the panel of altered miRNAs. Analysis of miRNAs for mRNA targets using TargetScan 7.1 was restricted to those above two fold induction (dashed line) ( B ) Tables showing the list of those miRNAs showing at least a two fold increase or decrease following KChIP2 silencing. ( C ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from rat, showing hybridization of the seed region. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A and SCN1B while two sites exist for KCND3. ( D ) Real-time qPCR analysis showing percent change of miR-34b/c expression from control cells in NRVM transfected with KChIP2.3 (n = 5), KChIP2.6 (n = 6), KChIP2.4 (n = 4), or KChIP2 siRNA (n = 4–5). ( E ) Cytosolic, membrane, and nuclear fractions of native adult rat heart tissue. KChIP2 nuclear localization was assessed by using lactate dehydrogenase (LDH), Serca2a, and Lamin-B as cytoplasmic, membrane, and nuclear markers respectively. ( F ) Representative z-stack images of adult rat ventricular myocyte. Nuclear stained regions (DAPI, blue) show the absence of cytosolic protein LDH (green), while KChIP2 (red) staining reveals significant colocalization. Data presented as mean ± SEM. *p<0.05; **p<0.01, compared to control. DOI: http://dx.doi.org/10.7554/eLife.17304.003

Article Snippet: 20–30 μg of protein extracts were loaded into SDS-PAGE gels, transferred to nitrocellulose membranes, and western blotting performed using lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:1000) to represent the cytosolic fraction, Lamin-B1 (Abcam Cat# ab16048 RRID: AB_443298 , 1:1000) representing the nuclear fraction, Serca2a (1:1000, Dr. Periasamy, Ohio State University) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) to observe localization.

Techniques: Microarray, Expressing, Hybridization, Sequencing, Transfection, Staining

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) A region from −500 to −191 of the miR-34b/c promoter was cloned into the promoterless luciferase construct, pGL4.10. This construct was co-transfected into COS-7 cells in the presence of KChIP2.3 (n = 3), KChIP2.6 (n = 8), or KChIP2.3 (n = 3) and compared to GFP alone. Renillin (pGL4.74) was used as a normalization control. Results are depicted as a % change in activity compared to GFP alone. ( B ) IgG and KChIP2 ChIP-PCR conducted on native adult rat cardiomyocytes. The target primer site residing within the cloned promoter was evaluated for enrichment following pull down (n = 3), showing significant enrichment of the target region. ( C ) Luciferase assay conducted in COS-7 cells to evaluate the outcome of deleting the putative DRE site in the miR-34b/c promoter. COS-7 cells were transfected with the same WT reporter construct inserted into the pGL4.10 vector or with the DRE motif deleted, both in the presence of KChIP2.6. Activity was normalized to renillin (pGL4.74). Deletion of a putative KChIP2 interaction site (DRE motif) partially abolished the repressive effect KChIP2.6 had over the miR-34b/c promoter (n = 4) compared to WT (n = 9). ( D ) COS-7 cells transfected with KChIP2.6 and the pGL4.10 containing the WT miR-34b/c promoter were treated with or without 10 mM caffeine for 6 hr, leading to promoter activation (n = 4). Results were normalized to renillin activity. Data presented as mean ± SEM. *p<0.05; **p<0.01, as indicated or compared to control. DOI: http://dx.doi.org/10.7554/eLife.17304.004

Article Snippet: 20–30 μg of protein extracts were loaded into SDS-PAGE gels, transferred to nitrocellulose membranes, and western blotting performed using lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:1000) to represent the cytosolic fraction, Lamin-B1 (Abcam Cat# ab16048 RRID: AB_443298 , 1:1000) representing the nuclear fraction, Serca2a (1:1000, Dr. Periasamy, Ohio State University) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) to observe localization.

Techniques: Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Plasmid Preparation, Activation Assay

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) Real-time qPCR evaluation of relative kcnip2 following treatment with 100 μM PE for 48 hr in NRVM (n = 6). Results normalized to ribosomal protein RPL27. ( B ) Evaluation of miR-34b (n = 8) and miR-34c (n = 7) relative expression in NRVM under control (no PE with Ad.GFP), 100 μM PE with Ad.GFP, or 100 μM PE with Ad.KChIP2 to maintain KChIP2 expression during the 48 hr treatment. Expression levels were normalized to small nucleolar RNA, U87. ( C ) The same treatment conditions in ( B ), evaluating relative mRNA expression for SCN5A (n = 10), SCN1B (n = 10), and KCND3 (n = 7). ( D ) Functional current-voltage measurements of I Na from NRVM under control (n = 29), PE+Ad.GFP (n = 27), and PE+Ad.KChIP2 (n = 30). ( E ) I/V curve for I to,f recordings in control (n = 7), PE+Ad.GFP (n = 9) and PE+Ad.KChIP2 (n = 9). Data presented as mean ± SEM. *p<0.05, **p<0.01, as indicated or compared to control, #p<0.05, compared to PE+Ad.GFP. DOI: http://dx.doi.org/10.7554/eLife.17304.007

Article Snippet: 20–30 μg of protein extracts were loaded into SDS-PAGE gels, transferred to nitrocellulose membranes, and western blotting performed using lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:1000) to represent the cytosolic fraction, Lamin-B1 (Abcam Cat# ab16048 RRID: AB_443298 , 1:1000) representing the nuclear fraction, Serca2a (1:1000, Dr. Periasamy, Ohio State University) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) to observe localization.

Techniques: Expressing, Functional Assay

Journal: eLife

Article Title: KChIP2 is a core transcriptional regulator of cardiac excitability

doi: 10.7554/eLife.17304

Figure Lengend Snippet: ( A ) Human tissue taken from the left ventricle of non-failing (NF) (n = 8) and failing patients (n = 20) evaluating KChIP2 and miR-34b/c RNA expression. KChIP2 levels were normalized to GAPDH and miR expression to small nucleolar RNA U6. ( B ) Evaluation of the human miR-34b/c reveals a conserved DRE motif in proximity of the miR-34b stem loop (−242 bp), as predicted by MatInspector, suggesting conservation of KChIP2 activity in the regulation of miR-34b/c expression. ( C ) Human heart failure tissue evaluating RNA levels for SCN5A , SCN1B , and KCND3 . Significant reductions in heart failure samples (n = 20) were observed for SCN5A and KCND3 , but not for SCN1B , compared to non-failing tissue (n = 8). ( D ) Alignment of the 3’-UTR of SCN5A, SCN1B, and KCND3 genes with miRs-34b/c from human. Grayed letters indicate variation in sequence between miR-34b and −34c. A single site of interaction is indicated for SCN5A, matching observations in the rat, while KCND3 has three potential sites, compared to two observed in the rat. Notably, SCN1B miR-34b/c targeting is not conserved in human shown by imperfect hybridization in the seed region. Data presented as mean ± SEM. *p<0.05; **p<0.01, as indicated or compared to control. #p<0.05, ## PP <0.01 compared to PE+Ad.GFP. DOI: http://dx.doi.org/10.7554/eLife.17304.008

Article Snippet: 20–30 μg of protein extracts were loaded into SDS-PAGE gels, transferred to nitrocellulose membranes, and western blotting performed using lactate dehydrogenase (Abcam Cat# ab52488 RRID: AB_2134961 , 1:1000) to represent the cytosolic fraction, Lamin-B1 (Abcam Cat# ab16048 RRID: AB_443298 , 1:1000) representing the nuclear fraction, Serca2a (1:1000, Dr. Periasamy, Ohio State University) and KChIP2 (UC Davis/NIH NeuroMab Facility Cat# 75–004 RRID: AB_2280942 , 1:50) to observe localization.

Techniques: RNA Expression, Expressing, Activity Assay, Sequencing, Hybridization