k252a  (Alomone Labs)

Journal: The Journal of Biological Chemistry

Article Title: Molecular Determinants of Kv1.3 Potassium Channels-induced Proliferation *

doi: 10.1074/jbc.M115.678995

Figure Lengend Snippet: Characterization of the Kv1.3 and Kv1.5 mutant channels containing the YS segment. A , average normalized activation and inactivation curves are shown as conductance-voltage relationships for Kv1.3, Kv1.5, the truncated Kv1.3-YS channel, and the chimeras Kv1.5-YS 532 and Kv1.5-YS 613 . All datasets were fitted to Boltzmann functions. Each data point is the mean ± S.E. of 6–11 cells. B , confocal images of non-permeabilized cells transfected with Kv1.3-YS-Cherry, Kv1.5-YS 532 -EGFP, and Kv1.5-YS 613 -EGFP. An extracellular anti-Kv1.3 antibody was used to label Kv1.3-YS ( green ), whereas the extracellular anti-Kv1.5 antibody was used for Kv1.5-YS 532 and Kv1.5-YS 613 chimeras ( red ). Nuclei were stained by Hoechst ( blue ). C , proliferation rate of the indicated channels or GFP-transfected cells (control) was determined by measuring EdU incorporation. Significant differences when comparing to Kv1.3 (*) or to control (#) are indicated. Statistical analysis was performed with one-way ANOVA followed by a Tukey's HSD multiple comparison. Each bar is the average of 9–15 determinations from 5 different assays. D , the average peak current amplitude obtained in cell-attached experiments for Kv1.5 channels and all the Kv1.5 chimeras was plotted against the % of the channels expressed at the plasma membrane ( upper graph ) or their normalized effect on proliferation (taking 100% as the proliferation rate of GFP-transfected HEK cells, lower graph ). The correlation between expression and current was fit to a linear regression curve ( y = 18.54 + 0.0066x, R 2 = 0.85, p = 0.008), but there was no correlation between proliferation and current amplitude ( R 2 = 0.23, p = 0.19).

Article Snippet: Non-permeabilized cells were incubated with anti-Kv1.3 or anti Kv1.5 extracellular primary antibodies (APC101 or APC150, Alomone Labs), whereas permeabilized cells were incubated with anti-Kv1.3 COOH (75-009, NeuroMab) or anti-Kv1.5 COOH (APC004, Alomone Labs), all at a final concentration of 1:50.

Techniques: Mutagenesis, Activation Assay, Transfection, Staining, Expressing

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Mutant KCNA5 is located in perinuclear packets and not on the cell surface of transfected HEK-293 cells and human (h)PASMC. A : HEK-293 cells transfected with WT KCNA5 ( a and c ) or G182R ( b and d ) were stained with anti-KCNA5 antibody (Ab-KCNA5, red) and

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Mutagenesis, Transfection, Staining

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: G182R and E211D mutations cause incomplete processing of KCNA5 in HEK-293 cells. HEK-293 cells were transfected with WT KCNA5, G182R, E211D, or G182R/E211D and subjected to standard immunoblot procedures. A : representative immunoblot from cells transfected

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Mutant KCNA5 forms functional homotetrameric channels. A : COS-1, HEK-293, and human pulmonary artery smooth muscle cells (PASMC) were transiently transfected with either empty vector [green fluorescent protein (GFP)] or WT KCNA5 (WT) as indicated. Whole

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Mutagenesis, Functional Assay, Transfection, Plasmid Preparation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Mutations in KCNA5 at G182 and E211 do not affect the pharmacological effect of 4-aminopyridine (4-AP). A : a standard I-V pulse protocol was delivered to HEK-293 cells transiently transfected with the indicated vector [WT KCNA5 ( a ), G182R ( b ), E211D (

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Transfection, Plasmid Preparation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Decreased G182R expression in COS-1 cells cannot be rescued by overexpression of K V β subunits. A , a : HEK-293 cells were transfected with WT KCNA5 or K V β1.3-HA alone or cotransfected with WT KCNA5, G182R, E211D, or G182R/E211D and K V β1.3-HA.

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Expressing, Over Expression, Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Two nonsynonymous mutations identified in the KCNA5 gene from idiopathic pulmonary arterial hypertension (IPAH) patients localize to the NH 2 -terminal tetramerization domain (T1 domain). A , left : schematic diagram of voltage-gated K + (K V ) channel subunit

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques:

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Cotransfection of K V β subunits affects KCNA5 channel kinetics. HEK-293 cells were transfected with WT KCNA5 alone (KCNA5) or in the presence of K V β1.3-hemagglutinin (HA) (KCNA5/K V β1.3). A and B : representative current recordings

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Cotransfection, Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: Voltage-dependent inactivation is accelerated in the G182R mutant KCNA5 channel. A standard 2-pulse inactivation protocol was used to determine channel availability after a 10-s prepulse in HEK-293 cells transiently transfected with WT KCNA5, G182R, E211D,

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Mutagenesis, Transfection

Journal: American Journal of Physiology - Cell Physiology

Article Title: Tetramerization domain mutations in KCNA5 affect channel kinetics and cause abnormal trafficking patterns

doi: 10.1152/ajpcell.00464.2009

Figure Lengend Snippet: G182R protein expression is significantly decreased in COS-1 cells. A : COS-1 cells were transiently transfected with water (Mock), WT-KCNA5, G182R, E211D, or G182R/E211D. Representative images are shown at ×40 magnification. B : transfected cells

Article Snippet: After cells were fixed in 4% paraformaldehyde-PBS for 15 min at room temperature, coverslips were incubated in blocking solution (2% BSA, 2% FBS, 0.1% Triton X-100 in PBS) for 1 h. Coverslips were then exposed to rabbit anti-KV1.5 antibody (Alomone Labs) at a dilution of 1:100 in blocking solution for 2 h at room temperature.

Techniques: Expressing, Transfection

Journal: The Journal of Neuroscience

Article Title: K+ Channel Kv3.4 Is Essential for Axon Growth by Limiting the Influx of Ca2+ into Growth Cones

doi: 10.1523/JNEUROSCI.1076-16.2017

Figure Lengend Snippet: Kv3.4 in the axonal growth cones of dorsal spinal commissural neurons. A–F , Transverse sections of the spinal cord of chick embryos were immunostained for Kv3.4. A , Absence of Kv3.4-IR in the dorsal spinal cord at HH17. Kv3.4-IR in precrossing commissural axons ( B–F , arrowheads) is evident during HH19-HH25 but disappears at HH27. D , Arrows indicate postcrossing commissural axons projecting from the other side of spinal cord. FP, Floor plate. G–L , Transverse sections of the spinal cord at HH23 were immunostained as indicated. G , Absence of Kv1.5-IR. H , Kv4.2-IR in the somata and dendrites of motoneurons (MN). I , Kv4.3-IR in the bifurcation zone (BZ). In addition to the BZ, Kv3.1b-IR is strong in postcrossing commissural axons ( J , arrow) but weak in precrossing commissural axons ( J , arrowhead). K , Absence of Kv3.2-IR. L , Kv3.3 in motoneurons. M–M″ , Double staining in transverse sections of the spinal cord at HH21 shows colocalization of Kv3.4 and axonin-1 in the growth cones (arrowheads) of commissural axons. N–N″ , Colocalization of Kv3.4 and axonin-1 in cultured dorsal spinal neurons isolated from HH21-HH23 chick embryos. O–P″ , Red fluorescence-tagged phalloidin colabeling reveals enrichment of Kv3.4 in the growth cone ( O–O″ ) and Kv3.1b in the soma/axon shaft ( P–P″ ) of cultured dorsal spinal neurons. Q–Q″ , Kv3.4 and DiI colabeling. White represents Kv3.4-abundant regions. Blue represents Kv3.4-sparse regions ( Q″ ). R , Ratio of Kv3.4/DiI in the soma, axon shaft, or growth cone of each neuron was obtained by dividing the fluorescence intensity of Kv3.4 by that of DiI. Data are mean ± SEM ( n = 8 neurons, pooled from three independent experiments done on different days). *** p

Article Snippet: After wash in low-salt Tris-buffered saline (LTBS; 25 m m Tris, 0.85% NaCl, pH 7.5) and then LTBS containing 0.1% Triton X-100 (LTBST), sections on slides were treated with 0.2% hydrogen peroxide in LTBS for 30 min. Nonspecific binding was blocked by 3% normal donkey serum and 2% BSA in LTBST for 1.5 h. Primary antibodies included rabbit anti-Kv1.5 (1:100), rabbit anti-Kv3.1b (1:100), rabbit anti-Kv3.2 (1:100), rabbit anti-Kv3.3 (1:100), rabbit anti-Kv3.4 (1:100), rabbit anti-Kv4.2 (1:250), and rabbit anti-Kv4.3 (1:1500).

Techniques: Double Staining, Cell Culture, Isolation, Fluorescence

Journal: The Journal of Physiology

Article Title: Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

doi: 10.1113/jphysiol.2007.134809

Figure Lengend Snippet: Some Kv1.5 channel subunits are localized in lipid raft fractions in adult rat atrial myocytes Western blot analysis of the distribution of Kv1.5 subunits, connexin-43 and caveolin-3 on step sucrose gradient of proteins from atrial myocardium.

Article Snippet: Then slides were incubated with rabbit anti-Kv1.5 polyclonal antibodies (pAbs) (1: 20), with mouse anti-caveolin-3 monoclonal antibodies (mAbs) (1: 50), with a mixture of mouse anti-caveolin-3 and rabbit anti-connexin-43 (1: 50) antibodies, or with a mixture of mouse anti-α-actinin sarcomeric and rabbit anti-Kv1.5 antibodies for 1 h at RT.

Techniques: Western Blot

Journal: The Journal of Physiology

Article Title: Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

doi: 10.1113/jphysiol.2007.134809

Figure Lengend Snippet: Kv1.5 do not co-localized with caveolin-3 in adult atrial tissue Immunolocalization of Kv1.5 subunits ( A ) and caveolin-3 ( B ) in cryosections of atrial myocardium. Double immunostaining of connexin-43 (FITC, C ) and caveolin-3 (Texas Red, D ) in cryosections of atrial myocardium. E , merged image of the same area of the section in C and D , showing the lack of overlap between connexin-43 and caveolin-3 stainings.

Article Snippet: Then slides were incubated with rabbit anti-Kv1.5 polyclonal antibodies (pAbs) (1: 20), with mouse anti-caveolin-3 monoclonal antibodies (mAbs) (1: 50), with a mixture of mouse anti-caveolin-3 and rabbit anti-connexin-43 (1: 50) antibodies, or with a mixture of mouse anti-α-actinin sarcomeric and rabbit anti-Kv1.5 antibodies for 1 h at RT.

Techniques: Double Immunostaining

Journal: The Journal of Physiology

Article Title: Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

doi: 10.1113/jphysiol.2007.134809

Figure Lengend Snippet: Surface expression of Kv1.5 subunits in neonatal cardiomyocytes A , in live cardiomyocytes, transfected GFP-tagged Kv1.5 subunits are clustered at the membrane surface adjacent to the bottom of laminin-coated glass support, as shown in the projection of Z sections in the lower panel. In contrast, GFP alone was homogeneously distributed in cardiomyocytes (inset). B , after the application of 2% MCD, clusters increased in size and were redistributed throughout the plasma membrane. C , bar graphs summarizing changes in cluster size upon MCD exposures; data are from 21 cardiomyocytes in control, and following incubation with 2% MCD for 7 min and 1 h 30 min. ** P

Article Snippet: Then slides were incubated with rabbit anti-Kv1.5 polyclonal antibodies (pAbs) (1: 20), with mouse anti-caveolin-3 monoclonal antibodies (mAbs) (1: 50), with a mixture of mouse anti-caveolin-3 and rabbit anti-connexin-43 (1: 50) antibodies, or with a mixture of mouse anti-α-actinin sarcomeric and rabbit anti-Kv1.5 antibodies for 1 h at RT.

Techniques: Expressing, Transfection, Incubation

Journal: The Journal of Physiology

Article Title: Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

doi: 10.1113/jphysiol.2007.134809

Figure Lengend Snippet: Effect of cholesterol depletion on outward current parameters resulting from Kv1.5 subunit overexpression in neonatal cardiomyocytes Current density–voltage relationships ( A ) and voltage dependence ( B ) of I Kur activation under control conditions (•) and following 7 min MCD application (^). In A and B , each point represents average data from 5 cells.

Article Snippet: Then slides were incubated with rabbit anti-Kv1.5 polyclonal antibodies (pAbs) (1: 20), with mouse anti-caveolin-3 monoclonal antibodies (mAbs) (1: 50), with a mixture of mouse anti-caveolin-3 and rabbit anti-connexin-43 (1: 50) antibodies, or with a mixture of mouse anti-α-actinin sarcomeric and rabbit anti-Kv1.5 antibodies for 1 h at RT.

Techniques: Over Expression, Activation Assay