Journal: Aging (Albany NY)

Article Title: Constructing a novel prognostic model for triple-negative breast cancer based on genes associated with vasculogenic mimicry

doi: 10.18632/aging.205806

Figure Lengend Snippet: Validation of biomarkers. ( A ) The mRNA expression of KCND2 and VSTM4 in 20 pairs of tissues, including TNBC adjacent cancer and tumor tissues. ( B ) The protein expression of KCND2 was verified in cell lines. ( C ) The expression of KCND2 in the TNBC adjacent cancer and tumor tissues was verified by immunohistochemistry. NS, no significance; ***P < 0.001.

Article Snippet: Antibodies to KCND2 (21298-1-AP), were purchased from Proteintech (Wuhan, China).

Techniques: Biomarker Discovery, Expressing, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: Constructing a novel prognostic model for triple-negative breast cancer based on genes associated with vasculogenic mimicry

doi: 10.18632/aging.205806

Figure Lengend Snippet: Depletion of KCND2 and VSTM4 reduces the proliferation and migration of TNBC cells in vitro . ( A ) The knockdown efficiency of siRNA targeting KCND2 in BT-549 cells was verified. ( B ) EdU incorporation analysis and ( C ) CCK-8 assay revealed that the knockdown of KCND2 affected the cell proliferation in BT-549 cells. ( D ) The knockdown efficiency of siRNA targeting VSTM4 in MDA-MB-231 cells was verified. ( E ) EdU incorporation analysis and ( F ) CCK-8 assay revealed that the knockdown of VSTM4 impacts cell proliferation in MDA-MB-231 cells. ( G ) The wound healing assay demonstrated that the knockdown of KCND2 affected the migration of BT-549 cells. ( H ) Wound healing assay results showed that the knockdown of VSTM4 impacted the migration of MDA-MB-231 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies to KCND2 (21298-1-AP), were purchased from Proteintech (Wuhan, China).

Techniques: Migration, In Vitro, Knockdown, CCK-8 Assay, Wound Healing Assay

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: KChIP2c regulates Kv4.2 channel current density and kinetics. a and b, experimental configuration in which Kv4.2 is expressed alone (gray) or coexpressed with KChIP2c (yellow). This color scheme is used throughout Fig. 1. c, voltage-clamp recording configuration for measuring Kv4.2 K+ currents. d, representative peak outward K+ current mediated by Kv4.2 and evoked with a step depolarization. e, peak outward K+ current density. Raw current was normalized to cell size measured by whole-cell capacitance on cell break-in. f, time required for the outward K+ current to rise from 10 to 90% of the peak. g and h, kinetics of channel inactivation was measured by fitting the falling phase of peak outward K+ current with a double-exponential decay and comparing fast (g) or slow (h) components across conditions. i, normalized conductance and current versus applied test membrane potential for VDA and VDI protocols. Colored connecting lines represent Boltzman curve fits. j, time-dependent fractional recovery of peak outward K+ current after a VDI prepulse. Curves are fitted to a single exponential function. Error bars represent mean ± S.E. *, p < 0.05; ****, p < 0.0001 by unpaired t test. Refer to Tables 1 and ​and22 for numerical data and replicate information.

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques:

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: Biophysical properties of Kv4 family members in nominal 0 μm free Ca2+ (gray boxes) or ∼10 μm free Ca2+ (red boxes)

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques:

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: Comparison of biophysical properties between Kv4.2-KChIP2 isoform complexes in nominal 0 μm free Ca2+ (gray) or 10 μm free Ca2+ (red)

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques:

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: Intracellular Ca2+ regulates Kv4.2 channel current density. a, theoretical free intracellular Ca2+ concentration at varying ratios of Ca2+ and buffer. Free Ca2+ was calculated using webmaxchelator (https://web.stanford.edu/∼cpatton/webmaxcS.htm) (Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.). b, schematic describing intracellular dialysis of buffering solutions to clamp free Ca2+. Color scheme is used throughout Figs. 2​2​–5 for nominal 0 μm free Ca2+ (black/white) and ∼10 μm free Ca2+ (red). c–e, construct expression (top), VDA protocol (above-middle inset), representative VDA K+ currents (above-middle), VDA current density versus test potential (below-middle), and normalized conductance and current versus applied test membrane potential for VDA and VDI protocols (bottom, Kv4.2 data also appears in Fig. 4b). Curves were fitted to a Boltzmann function. Horizontal scale bar, 100 ms; vertical scale bars, 1 nA. Error bars represent mean ± S.E. **, p < 0.01 by unpaired t test. Refer to Tables 1 and ​and22 for numerical data and replicate information.

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques: Concentration Assay, Construct, Expressing

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: Intracellular Ca2+ regulates Kv4.2 peak current. a, representative evoked outward K+ current in HEK293T cells expressing Kv4.2 and KChIP2c either in nominal 0 μm Ca2+ (black trace) or ∼10 μm free Ca2+ (red trace). b, peak outward K+ current density. Raw current was normalized to cell size measured by whole-cell capacitance on cell break-in. c, time required for the outward K+ current to rise from 10 to 90% of the peak. d and e, kinetics of channel inactivation was measured by fitting the falling phase of peak outward K+ current with a double-exponential decay and comparing fast (d) or slow (e) components across conditions. Error bars, mean ± S.E. *, p < 0.05; **, p < 0.01 by unpaired t test. Refer to Table 2 for numerical data and replicate information.

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques: Expressing

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 is conserved across the Kv4 family. a, sequence alignment of the intracellular N-terminal domains of Kv4 family members. Proposed KChIP-binding domains (KChIP BD 1/2) and the Kv4 tetramerization domain (T1) are shown. b, normalized conductance and current versus applied test membrane potential for VDA and VDI protocols in Kv4.1, Kv4.2 (data also appears in Fig. 2d), or Kv4.3 and KChIP2c expressing HEK293T cells are shown. c, because of day-to-day variability of Kv4.1 and Kv4.3 construct expression, current density (pA/picofarad (pF)) was normalized by day of experiment. Statistical analysis by two-way ANOVA showed a significant effect of treatment (p < 0.0001) but not by Kv4 isoform expression (due to normalization) or the interaction (p = 0.9076). Sidak's multiple comparisons among the Kv4 isoforms in control and treated conditions yielded significant differences within groups (Kv4.1, p = 0.0026; Kv4.2, p = 0.0107; and Kv4.3, p = 0.0253). d and e, macroscopic channel kinetics are presented as described in Fig. 3. d, statistical analysis of 10–90% rise times found significant differences among Kv4 isoforms (p = 0.0041) but not by treatment (p = 0.1735) or the interaction (p = 0.8888). Comparison of control and 10 μm Ca2+ 10–90% rise time within Kv4 isoforms revealed no differences (Kv4.1, p = 0.9626; Kv4.2, p = 0.5717; and Kv4.3, p = 0.8137). e, significant differences in the variation between treatment (p = 0.0320), Kv4 isoform expression (p < 0.0001), and the interaction (p = 0.0080) were found for the fast component of inactivation (τ1). Among the Kv4 isoforms, only Kv4.2 showed a significant Ca2+-dependent acceleration of τ1 (Kv4.1, p = 0.9237; Kv4.2, p = 0.0004; and Kv4.3, p = 0.9373). f, statistical tests showed no significant variation by treatment (p = 0.9091) or the interaction (p = 0.5518), whereas Kv4 isoform expression (p = 0.0053) affected the slow component of inactivation (τ2). However, Ca2+ did not regulate τ2 of any Kv4 isoform (Kv4.1, p = 0.9088; Kv4.2, p = 0.9745; and Kv4.3, p = 0.7846). Error bars represent mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by two-way ANOVA and Sidak's multiple comparison test. Refer to Table 1 for numerical data and replicate information.

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques: Sequencing, Binding Assay, Expressing, Construct

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: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques: Sequencing, Mutagenesis, Construct, Expressing

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: Biophysical properties of Kv4.2–KChIP1, -3, and -4 isoform complexes in 0 μm free Ca2+ (gray) or ∼10 μm free Ca2+ (red)

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques:

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: Model for Ca2+ regulation of Kv4–KChIP complexes. a, variable N-terminal domain of Ca2+-resistant KChIP–Kv4 complexes share a conserved polybasic motif. b, Ca2+ regulates a subset of KChIP isoforms by promoting Kv4 channel current density.

Article Snippet: Heterologous Kv4 channel expression was carried out using human Kv4.1 (RC207353), Kv4.2 (RC215266), and Kv4.3 (RC219447) (Origene).

Techniques:

Journal: Frontiers in Neuroanatomy

Article Title: Calcium/calmodulin-dependent protein kinase II associates with the K + channel isoform Kv4.3 in adult rat optic nerve

doi: 10.3389/fnana.2022.958986

Figure Lengend Snippet: Kv4.3 immunopositivity in optic nerve axons. Confocally imaged portion of optic nerve that was longitudinally sectioned and immunostained with antibodies directed against Kv4.3 (upper panel) and NF-70 (middle panel). The upper and middle panels are superimposed in the lower panel (“merge”). The tip of each arrowhead points to an axon (based on NF-70 immunopositivity). Arrowheads of a given color point at the same axon. Different colors are used to point at different axons. Otherwise, the color and direction of the arrowheads (upward or downward) are arbitrary. The calibration bar in the lower panel shows 10 μm and applies to all panels.

Article Snippet: The Kv4.3 blocking peptide (Alomone, BLP-PC017) is a synthetic peptide corresponding to amino acid residues 451–468 of human Kv4.3.

Techniques:

Journal: Frontiers in Neuroanatomy

Article Title: Calcium/calmodulin-dependent protein kinase II associates with the K + channel isoform Kv4.3 in adult rat optic nerve

doi: 10.3389/fnana.2022.958986

Figure Lengend Snippet: Control for non-specific binding by rabbit anti-Kv4.3 antibody. (A) Fields of optic nerve that were longitudinally sectioned and processed as in , except that the anti-Kv4.3 antibody in the upper panel was preincubated with immunogen, and the anti-Kv4.3 antibody in the lower panel was not (as in ). These fields were imaged at identical confocal microscope settings (laser intensity, photomultiplier gain, and pinhole diameter). The calibration bar in the lower panel shows 10 μm and applies to both panels. (B) Western-blotted lanes of optic nerve lysate. The lanes were processed identically except that the anti-Kv4.3 antibody was either preincubated with immunogen (in the lane marked “+immunogen”) or it was used without this preincubation (in the lane marked “–immunogen”). To gauge protein load, both lanes were probed with rat anti-myelin basic protein antibody. The fluorescence of the anti-rabbit secondary antibody is pseudo-colored red. The fluorescence of the anti-rat secondary antibody is pseudo-colored blue. The molecular weight and migration distance of protein standards that were run in an adjacent lane are shown along the right edge.

Article Snippet: The Kv4.3 blocking peptide (Alomone, BLP-PC017) is a synthetic peptide corresponding to amino acid residues 451–468 of human Kv4.3.

Techniques: Binding Assay, Microscopy, Western Blot, Fluorescence, Molecular Weight, Migration

Journal: Frontiers in Neuroanatomy

Article Title: Calcium/calmodulin-dependent protein kinase II associates with the K + channel isoform Kv4.3 in adult rat optic nerve

doi: 10.3389/fnana.2022.958986

Figure Lengend Snippet: Reciprocal co-immunoprecipitation of Kv4.3 and CaMKII. Western-blotted lanes (1-7), with one lane cut vertically into two parts (lanes 3a, 3b). The material that was loaded into the gel lanes for electrophoresis, and the antibody used to probe each Western-blotted lane, are listed above and below the lanes, respectively. The lanes were loaded with either optic nerve lysate (lanes 3a, 3b, 6), elute from the anti-Kv4.3 antibody pull-down (lanes 1, 5), or elute from the anti-CaMKII antibody pull-down (lanes 2, 4, 7). The Western-blotted lanes were probed with rabbit anti-Kv4.3 antibody (“Kv4.3”) and anti-rabbit secondary antibody, mouse anti-CaMKII antibody (“CaMKII”) and anti-mouse secondary antibody, or anti-rabbit secondary antibody without primary antibody (“2° only”). The molecular weight and migration distance of protein standards that were run in an adjacent lane are shown along the left edge. Note that the faint bands around 60 kDa in lane 3a are absent in lane 3b.

Article Snippet: The Kv4.3 blocking peptide (Alomone, BLP-PC017) is a synthetic peptide corresponding to amino acid residues 451–468 of human Kv4.3.

Techniques: Immunoprecipitation, Western Blot, Electrophoresis, Molecular Weight, Migration