Journal: PLoS ONE

Article Title: NHE Isoform Switching and KChIP2 Upregulation in Aging Porcine Atria

doi: 10.1371/journal.pone.0082951

Figure Lengend Snippet: Transcripts showing statistically most significant LA expression differences between young and old swine.

Article Snippet: Supernatants (50 µg protein/lane) were heated to 45°C and size-fractionated on 4–12% Bis-Tris gels (Invitrogen, NY, USA), transferred electrophoretically onto nitrocellulose membranes (VWR) and probed with mouse anti-KChIP2 K60/73 (UC Davis/NIH NeuroMab Facility) and mouse anti-GAPDH (Sigma) antibodies.

Techniques: Expressing

Journal: PLoS ONE

Article Title: NHE Isoform Switching and KChIP2 Upregulation in Aging Porcine Atria

doi: 10.1371/journal.pone.0082951

Figure Lengend Snippet: A. Mean KChIP2 ( KCNIP2 ) transcript expression quantified by real-time qPCR, standardized to GAPDH expression in each sample and then normalized to expression measured from a single young pig LA sample. **p<0.01, old v young; all others p>0.05; n = 3–5. B. Upper : mean cardiac KChIP2 protein expression visualized by western blotting, standardized by total protein for loading. Lower : GAPDH expression for comparison. Each lane is from a different individual. C. Mean cardiac KChIP2 protein expression quantified by band densitometry of blots as in panel F, normalized to GAPDH expression. *p<0.05 old v young; n = 3–4. D. In silico modeling data showing the predicted effects of 3.8-fold KChIP2 upregulation on human atrial myocyte I to ( lower ) and action potential morphology ( upper ).

Article Snippet: Supernatants (50 µg protein/lane) were heated to 45°C and size-fractionated on 4–12% Bis-Tris gels (Invitrogen, NY, USA), transferred electrophoretically onto nitrocellulose membranes (VWR) and probed with mouse anti-KChIP2 K60/73 (UC Davis/NIH NeuroMab Facility) and mouse anti-GAPDH (Sigma) antibodies.

Techniques: Expressing, Western Blot, In Silico

Journal: PLoS ONE

Article Title: Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit

doi: 10.1371/journal.pone.0115731

Figure Lengend Snippet: (A) Triple staining of the neuronal marker UCHL1 and two different TRPV1 antibodies derived from rabbit and goat, respectively, to facilitate the analysis of various targets. The staining intensities obtained with both TRPV1 antibodies correlated significantly (Spearmans ρ = 0.96, p<2.2e-16). (B-E, G) Co-labeling of TRPV1 and CART (B), Nos1 (C), KChIP1 (D), KChIP2 (E), and CaMKIIα (G). Plots of respective controls are shown in . (F) Average fluorescence intensities of TRPV1 and the indicated targets in TRPV1-negative (grey) and -positive (black) neurons. Signal intensities of all analyzed targets were significantly higher within the TRPV1(+) population (n = 3 with>3000 analyzed neurons per experiment, paired two-tailed t-tests).

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1∶2000, Novus, Cambridge, UK, #NB110-58872), rabbit polyclonal anti-TRPV1 (1∶1000, Alomone labs, Jerusalem, Israel, # ACC-030), goat polyclonal anti-TRPV1 (1∶500, R&D Systems, #AF3066), mouse monoclonal anti hCART, (1∶3000, R&D Systems, #MAB163), rabbit monoclonal anti NOS1 (1∶500, clone C7D7, Cell Signaling, Danvers, MA, #4231), mouse monoclonal anti CaMKIIα (1∶2000, clone 6G9, Thermo Scientific, #MA1-048), rabbit monoclonal anti CaMKIV (1∶500, Millipore, clone EP2564Y, #04-1081), mouse monoclonal anti KChIP1 (1∶500, Abcam, Cambridge, UK, #ab99013), mouse monoclonal anti KChIP2 (1∶1000, UC Davis/NIH NeuroMab Facility, Clone K60/73, #75-004), rabbit monoclonal anti phospho RIIα (S96) (1∶1000, clone 151, Abcam, # ab32390), mouse monoclonal anti phospho-p44/42 MAPK (T202/Y204) (1∶250, clone E10, Cell Signaling, #9106), rabbit monoclonal anti-Prostaglandin D Synthase (1∶1000, clone E12357, Abcam, ab182141), mouse monoclonal anti-NF200 (1∶2000, clone N52, Sigma-Aldrich, Munich, Germany, #N0142), highly cross-adsorbed Alexa 647, 594, and 488 conjugated secondary antibodies (Invitrogen, Carlsbad, CA).

Techniques: Staining, Marker, Derivative Assay, Labeling, Fluorescence, Two Tailed Test

Journal: eLife

Article Title: Mutant huntingtin enhances activation of dendritic Kv4 K + channels in striatal spiny projection neurons

doi: 10.7554/eLife.40818

Figure Lengend Snippet: ( a ) Ca 2+ transients in response to bAPs (arrows) at proximal and distal dendritic spines in iSPNs from BACHD mice with the internal perfusion of KChIP2 antibody (1:50); perfusion rescued dendritic excitability in HD iSPNs. ( b ) The dendritic index was significantly smaller with internal perfusion of the denatured KChIP2 antibody (boiled) (p=0.0022, Mann-Whitney U, Two-Tailed, n = 6). ( c ) Voltage responses to optogenetic stimulation of corticostriatal terminals at distal dendrites in iSPNs from BACHD mice with internal perfusion of KChIP2 antibody before and after 4-AP (2 mM). ( d ) The Kv4 index was significantly reduced by antibody perfusion (p=0.0006, Mann-Whitney U, Two-Tailed, n = 7). In all the dialysis experiments, recordings were taken >30 min after entering whole cell mode. ( e ) Western blot of Kv4.2 from striatum homogenates. ( f ) Kv4.2 protein levels show no significant difference between WT and Q175 ±mice (p=0.061, Mann-Whitney U, Two-Tailed, n = 5. ( g ) Co-immunoprecipitation of Cav3.2 channels with Kv4.2 channels from mouse striatum (Kv4.2 pulldown). ( h ) Voltage responses to optogenetic stimulation of corticostriatal terminals at distal dendrites of iSPNs from BACHD mice in the presence of the Cav3 Ca 2+ channel blocker mibefradil (1 μM) before and after 4-AP (2 mM). ( i ) Changes in the Kv4 index were consistent with the loss of dendritic excitability in iSPNs being mediated by the interaction of Cav3 Ca 2+ channels with Kv4.2 channels through KChIPs (p=0.0006, Mann-Whitney U, Two-Tailed, n = 7. See . 10.7554/eLife.40818.023 Figure 3—source data 1. Source data for .

Article Snippet: The KChIP2 antibody (Neuromab 75–004) also was diluted at the same 1:50 ratio.

Techniques: MANN-WHITNEY, Two Tailed Test, Western Blot, Immunoprecipitation

Journal: eLife

Article Title: Mutant huntingtin enhances activation of dendritic Kv4 K + channels in striatal spiny projection neurons

doi: 10.7554/eLife.40818

Figure Lengend Snippet: ( a ) KChIPs co-immunoprecipitated with Kv4.2 in mice striata, KChIP1 and KChIP2 more robustly associated with Kv4.2 than KChIP3/4. To the right is a schematic of the Kv4.2/KChIP/Cav3.2 membrane complex. ( b ) Detection of Kv4.2 phosphorylation at Thr607 and Thr602, P-Kv4.2 levels were normalized to total Kv4.2; in Q175 ±mice, Kv4.2 phosphorylation was decreased at both Thr607 (p=0.03078, Mann-Whitney U, Two-Tailed, n = 6) and Thr602 (p=0.0226, Mann-Whitney U, Two-Tailed, n = 6). ( c ) Co-immunoprecipitation of KChIP2 with Kv4.2 in WT striata after incubation with BDNF or with vehicle control; association of KChIP2 was decreased (p=0.01208, Mann-Whitney U, Two-Tailed, n = 5). ( d ) Co-immunoprecipitation of KChIP2 with Kv4.2 in Q175 ±striata after incubation with BDNF or with vehicle control; no difference in Kv4.2 association with KChIP2 (p=0.83366, Mann-Whitney U, Two-Tailed, n = 5). ( e ) Co-immunoprecipitation of KChIP2 with Kv4.2 in Q175 ±striata after incubation with ROCKi +BDNF or with vehicle control; association of KChIP2 with Kv4.2 was decreased (p=0.03662, Mann-Whitney U, Two-Tailed, n = 5). ( f ) Diagram of the proposed mechanism showing how TrkBR and p75NTR signaling modulate KChIP association with Kv4.2 and Kv4.2 channel gating. See . 10.7554/eLife.40818.027 Figure 4—source data 1. Source data for .

Article Snippet: The KChIP2 antibody (Neuromab 75–004) also was diluted at the same 1:50 ratio.

Techniques: Immunoprecipitation, MANN-WHITNEY, Two Tailed Test, Incubation

Journal: The Journal of Neuroscience

Article Title: K + Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control

doi: 10.1523/JNEUROSCI.1619-16.2017

Figure Lengend Snippet: Intrathecal injection of Kv4.3 ASO reduces all Kv4 complex components in the L4–L6 DRGs. Rats were intrathecally injected with Kv4.3 ASO twice daily for 3 consecutive days (D1–D3) and killed on day 4 (D4) or D7. Rats injected with vehicle (V) or LacZ ASO (Z) were used as controls. A, In the L5 DRG of rats treated with Kv4.3 ASO, Kv4.3-, KChIP1-, KChIP2-, and DPP10-IR were greatly reduced on D4 but returned to the normal levels on D7. Scale bar, 40 μm. B–E, Quantitative data of A. C, C7 DRG. L, L5 DRG. F–I, Western blotting was performed using total proteins (F, G) or plasma membrane proteins (H, I) isolated from the bilateral L4–L6 DRGs on D4. Kv4.3 ASO treatment reduces Kv4.3, KChIP1, and DPP10 in both protein preparations. Bands of GAPDH and cadherin were loading controls for total proteins and plasma membrane proteins, respectively. Data are mean ± SD (n = 3). **p < 0.01, compared with vehicle (normalized to 1; Student's t test). ***p < 0.001, compared with vehicle (normalized to 1; Student's t test).

Article Snippet: Nonspecific binding was blocked by 3% normal donkey serum plus 2% bovine serum albumin in LTBST for 1.5 h. Primary antibodies included rabbit anti-DPP10 (1:3000), rabbit anti-GFAP (1:8000; Dako catalog #Z0334, RRID:AB_2314535), rabbit anti-Iba1 (1:800; Wako catalog #019-19741, RRID:AB_839504), mouse anti-KChIP1 (1:100; NeuroMab catalog #75-003, RRID:AB_10673162), mouse anti-KChIP2 (1:50; NeuroMab catalog #75-004, RRID:AB_2280942), and mouse anti-Kv4.3 (1:4000; NeuroMab catalog #75-017, RRID:AB_2314723).

Techniques: Injection, Western Blot, Isolation

Journal: The Journal of Neuroscience

Article Title: K + Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control

doi: 10.1523/JNEUROSCI.1619-16.2017

Figure Lengend Snippet: KChIP1 or KChIP2 ASO reduces all Kv4 complex components in the DRGs and induces mechanical hypersensitivity. Rats were intrathecally injected with vehicle, LacZ ASO, KChIP1 ASO, or KChIP2 ASO during D1–D3. A, B, KChIP1 ASO induces bilateral mechanical hypersensitivity during D1–D5. Thermal hypersensitivity was not detected. L, Left side; R, right side. Data are mean ± SEM (n = 6). *p < 0.05, compared with the baseline (BL) on the corresponding side (Tukey's post hoc test after one-way ANOVA). **p < 0.05, compared with the baseline (BL) on the corresponding side (Tukey's post hoc test after one-way ANOVA). ***p < 0.001, compared with the baseline (BL) on the corresponding side (Tukey's post hoc test after one-way ANOVA). C–F, Rats were killed on D4 after ASO treatment. Western blotting was performed using total proteins (C, D) or plasma membrane proteins (E, F) isolated from the bilateral L4–L6 DRGs on D4. KChIP1 ASO treatment reduces Kv4.3, KChIP1, and DPP10 in both protein preparations. G, H, KChIP2 ASO induces bilateral mechanical hypersensitivity during D1–D5. Thermal hypersensitivity was not detected. **p < 0.05, compared with the baseline (BL) on the corresponding side by Tukey's post hoc test after one-way ANOVA. ***p < 0.001, compared with the baseline (BL) on the corresponding side by Tukey's post hoc test after one-way ANOVA. I–K, In rats treated with KChIP2 ASO, Kv4.3-, KChIP1- and KChIP2-IR in L5 DRG neurons were reduced on D4. L, In rats treated with KChIP1 ASO, KChIP2-IR in L5 DRG neurons was also reduced on D4. Data are mean ± SD (n = 3). *p < 0.05, compared with vehicle (normalized to 1; Student's t test). **p < 0.01, compared with vehicle (normalized to 1; Student's t test). ***p < 0.001, compared with vehicle (normalized to 1; Student's t test).

Article Snippet: Nonspecific binding was blocked by 3% normal donkey serum plus 2% bovine serum albumin in LTBST for 1.5 h. Primary antibodies included rabbit anti-DPP10 (1:3000), rabbit anti-GFAP (1:8000; Dako catalog #Z0334, RRID:AB_2314535), rabbit anti-Iba1 (1:800; Wako catalog #019-19741, RRID:AB_839504), mouse anti-KChIP1 (1:100; NeuroMab catalog #75-003, RRID:AB_10673162), mouse anti-KChIP2 (1:50; NeuroMab catalog #75-004, RRID:AB_2280942), and mouse anti-Kv4.3 (1:4000; NeuroMab catalog #75-017, RRID:AB_2314723).

Techniques: Injection, Western Blot, Isolation

Journal: The Journal of Neuroscience

Article Title: K + Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control

doi: 10.1523/JNEUROSCI.1619-16.2017

Figure Lengend Snippet: Intrathecal ASO injections do not affect KChIP1, KChIP2, and DPP10 expression in the spinal cord. Rats were intrathecally injected with ASO for LacZ (Z), Kv4.3, KChIP1, KChIP2, or DPP10 during D1–D3 and killed on D4. Sections of the L5 spinal cord segment were immunostained. A–C, KChIP1-, KChIP2-, or DPP10-IR in the dorsal spinal cord (on either side) is similar between each ASO group and the vehicle (V) group. Scale bar, 210 μm. D–F, Quantitative data of A–C. Data are mean ± SD (n = 3), compared with vehicle by Student's t test.

Article Snippet: Nonspecific binding was blocked by 3% normal donkey serum plus 2% bovine serum albumin in LTBST for 1.5 h. Primary antibodies included rabbit anti-DPP10 (1:3000), rabbit anti-GFAP (1:8000; Dako catalog #Z0334, RRID:AB_2314535), rabbit anti-Iba1 (1:800; Wako catalog #019-19741, RRID:AB_839504), mouse anti-KChIP1 (1:100; NeuroMab catalog #75-003, RRID:AB_10673162), mouse anti-KChIP2 (1:50; NeuroMab catalog #75-004, RRID:AB_2280942), and mouse anti-Kv4.3 (1:4000; NeuroMab catalog #75-017, RRID:AB_2314723).

Techniques: Expressing, Injection

Journal: The Journal of Neuroscience

Article Title: K + Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control

doi: 10.1523/JNEUROSCI.1619-16.2017

Figure Lengend Snippet: Detection of a Kv4.3/KChIP1/KChIP2/DPP10 complex in the DRG. Total proteins (Total), plasma membrane proteins (PM), and rough ER proteins (rER) were isolated from rat lumbar DRGs, respectively. A, Representative Western blot data show Kv4.3, KChIP1, KChIP2, and DPP10 in each protein preparation. Cadherin and calnexin are markers for the PM and rER, respectively. The purity of PM proteins was confirmed by the presence of cadherin and the absence of calnexin, whereas the purity of rER proteins was confirmed by the presence of calnexin and the absence of cadherin. B, C, In addition to immunoprecipitation of Kv4.3, rabbit anti-Kv4.3 antibody (α-Kv4.3) coimmunoprecipitates KChIP1, KChIP2, and DPP10 from the PM proteins (B) and rER proteins (C), respectively. Nonspecific rabbit IgG was used as a control of immunoprecipitation, and “extract” indicates a bead-only loading control.

Article Snippet: Nonspecific binding was blocked by 3% normal donkey serum plus 2% bovine serum albumin in LTBST for 1.5 h. Primary antibodies included rabbit anti-DPP10 (1:3000), rabbit anti-GFAP (1:8000; Dako catalog #Z0334, RRID:AB_2314535), rabbit anti-Iba1 (1:800; Wako catalog #019-19741, RRID:AB_839504), mouse anti-KChIP1 (1:100; NeuroMab catalog #75-003, RRID:AB_10673162), mouse anti-KChIP2 (1:50; NeuroMab catalog #75-004, RRID:AB_2280942), and mouse anti-Kv4.3 (1:4000; NeuroMab catalog #75-017, RRID:AB_2314723).

Techniques: Isolation, Western Blot, Immunoprecipitation