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: Molecular and cellular neurosciences

Article Title: A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking

doi: 10.1016/j.mcn.2019.06.007

Figure Lengend Snippet: Auxiliary subunits regulate BBS-Kv4.2 surface expression in HEK 293T cells. (A) Auxiliary subunits were shown to increase BBS-Kv4.2 membrane expression in HEK 293T cells via western blot analysis. Cells transfected with BBS-Kv4.2 alone or together with DPP6 or KChIP2 were processed for surface biotinylation. (B) Surface labeling experiments show that auxiliary subunits facilitate BBS-Kv4.2 membrane localization in HEK 293T cells. Cells transfected with BBS-Kv4.2 alone or together with DPP6 and KChIP2 were incubated with RhBTX at 17°C for 30 min. Cells were fixed, permeabilized and stained with anti-Myc antibody. Co-transfection with DPP6 and KChIP2 increased surface BBS-Kv4.2 expression. Scale bar: 10 μm. (C) Graphical representation of (B) and Figure S2. The surface stain intensity of S3-S4 BBS-Kv4.2 (BBS-Kv4.2–285) is significantly higher than that of S1-S2 BBS-Kv4.2 (BBS-Kv4.2–220). n = 15 cells for each group. ***p < 0.001 vs alone, #p < 0.05, ###p < 0.001 vs BBS-Kv4.2–220. (D) KChIP2 and DPP6 auxiliary subunits increase Kv4.2 and BBS-Kv4.2 current density. Left, Kv4.2 and BBS-Kv4.2 current traces. Vertical and horizontal scale bars correspond to 100 pA/pF and 100 ms respectively. Right, current density for each construct co-expressed with DPP6 or KChIP2. BBS-Kv4.2 alone exhibits a decreased current density compared to that of Kv4.2 but the current densities of both constructs are similarly increased by auxiliary subunits.

Article Snippet: Kv4.2 (K57/1): Neuromab 75–016, 1:100 for staining, 1:2000 for western blot; Myc: Millipore, 05–419, 1:500 for staining, 1:5000 for western blot; DPP6: Abcam, ab41811, 1:1000 for western blot; KChIP2: pan KChIP, Neuromab, 75–006, 1:1000 for western blot; Actin: Sigma, A-1978, 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse: Invitrogen, A-11029, 1:500; Alexa Fluor 680 goat anti-mouse: Invitrogen, A-21057, 1:10000; Alexa Fluor 680 goat anti-rabbit: Invitrogen, A-21076, 1:10000.

Techniques: Expressing, Western Blot, Transfection, Labeling, Incubation, Staining, Cotransfection, Construct

Journal: Molecular and cellular neurosciences

Article Title: A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking

doi: 10.1016/j.mcn.2019.06.007

Figure Lengend Snippet: Biophysical properties of Kv4.2 and BBS-Kv4.2 voltage gated K + currents.

Article Snippet: Kv4.2 (K57/1): Neuromab 75–016, 1:100 for staining, 1:2000 for western blot; Myc: Millipore, 05–419, 1:500 for staining, 1:5000 for western blot; DPP6: Abcam, ab41811, 1:1000 for western blot; KChIP2: pan KChIP, Neuromab, 75–006, 1:1000 for western blot; Actin: Sigma, A-1978, 1:10000 for western blot; Alexa Fluor 488 goat anti-mouse: Invitrogen, A-11029, 1:500; Alexa Fluor 680 goat anti-mouse: Invitrogen, A-21057, 1:10000; Alexa Fluor 680 goat anti-rabbit: Invitrogen, A-21076, 1:10000.

Techniques:

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