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kcne2 ab1  (Alomone Labs)


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    Alomone Labs kcne2 ab1
    Detecting <t>KCNE2</t> by 2 antibodies, <t>Ab1</t> and Ab2. A: transmembrane topology of KCNE2 and a pore-forming α-subunit. B: Ab1 and Ab2 epitopes in the human KCNE2 post-transmembrane domain (TMD) region (yellow shading in A). The human sequence is aligned with those of rat and guinea pig (GP); dot (.) denotes same residue as in the human sequence. Two potential phosphorylation sites in the Ab2 epitope region, Y96 and S98, are marked. C–F: probing KCNE2 expression in young adult (4 to 5 mo) spontaneously hypertensive rat (SHR) ventricular myocytes. SHR myocytes are cultured in serum-free medium for 3 days under the control conditions (control culture) or after overnight incubation with adenovirus carrying green fluorescent protein (Adv-GFP), HA-tagged KCNE2 [Adv-E2 (HA)], or a short hairpin sequence targeting rat KCNE2 nucleotides 319–340 (Adv-E2 si). C–E: immunoblots (IB) of whole cell lysates (WCL) from myocytes in control culture or incubated with adenovirus marked on top. Fresh in E is whole tissue lysate (WTL) prepared from freshly isolated SHR ventricular myocardium. The antibodies used for IB are marked below the images. In C, the central lane is split into 2 and probed with HA mAb (left half) and Ab1 (right half). Size marker positions are marked at left. Solid arrows point to the 2 major HA-tagged KCNE2 bands of 33 and 25 kDa, detected by HA mAb (red arrows; C), Ab1 (black arrows; C), and Ab2 (gray arrows; D). Open arrows point to unrelated proteins detected by HA mAb (C) and by Ab1 or Ab2 (E). Arrows with gradient shade point to the 33-kDa band seen in SHR ventricular myocytes not expressing HA-tagged KCNE2 (C, right, and E). *Highlights the 24-kDa band in WTL from freshly isolated myocardium that is missing in ventricular myocytes after culture under the control conditions (in E). Some lanes are cut and pasted together to avoid showing irrelevant lanes (marked by white stripes between pasted lanes). F: HA immunofluorescence (IF; stained with Alexa647) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence (nuclei) from a myocyte expressing HA-tagged KCNE2 (top) and a myocyte expressing GFP (bottom). In the former case, arrow and arrowhead point to HA IF on cell surface and in perinuclear region. In the latter case, there is no Alexa647 signal but strong GFP signal.
    Kcne2 Ab1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/blp-pc054/pmc03322735-261-5-12?v=Alomone+Labs
    Average 90 stars, based on 1 article reviews
    kcne2 ab1 - by Bioz Stars, 2026-07
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    1) Product Images from "KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner"

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00691.2011

    Detecting KCNE2 by 2 antibodies, Ab1 and Ab2. A: transmembrane topology of KCNE2 and a pore-forming α-subunit. B: Ab1 and Ab2 epitopes in the human KCNE2 post-transmembrane domain (TMD) region (yellow shading in A). The human sequence is aligned with those of rat and guinea pig (GP); dot (.) denotes same residue as in the human sequence. Two potential phosphorylation sites in the Ab2 epitope region, Y96 and S98, are marked. C–F: probing KCNE2 expression in young adult (4 to 5 mo) spontaneously hypertensive rat (SHR) ventricular myocytes. SHR myocytes are cultured in serum-free medium for 3 days under the control conditions (control culture) or after overnight incubation with adenovirus carrying green fluorescent protein (Adv-GFP), HA-tagged KCNE2 [Adv-E2 (HA)], or a short hairpin sequence targeting rat KCNE2 nucleotides 319–340 (Adv-E2 si). C–E: immunoblots (IB) of whole cell lysates (WCL) from myocytes in control culture or incubated with adenovirus marked on top. Fresh in E is whole tissue lysate (WTL) prepared from freshly isolated SHR ventricular myocardium. The antibodies used for IB are marked below the images. In C, the central lane is split into 2 and probed with HA mAb (left half) and Ab1 (right half). Size marker positions are marked at left. Solid arrows point to the 2 major HA-tagged KCNE2 bands of 33 and 25 kDa, detected by HA mAb (red arrows; C), Ab1 (black arrows; C), and Ab2 (gray arrows; D). Open arrows point to unrelated proteins detected by HA mAb (C) and by Ab1 or Ab2 (E). Arrows with gradient shade point to the 33-kDa band seen in SHR ventricular myocytes not expressing HA-tagged KCNE2 (C, right, and E). *Highlights the 24-kDa band in WTL from freshly isolated myocardium that is missing in ventricular myocytes after culture under the control conditions (in E). Some lanes are cut and pasted together to avoid showing irrelevant lanes (marked by white stripes between pasted lanes). F: HA immunofluorescence (IF; stained with Alexa647) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence (nuclei) from a myocyte expressing HA-tagged KCNE2 (top) and a myocyte expressing GFP (bottom). In the former case, arrow and arrowhead point to HA IF on cell surface and in perinuclear region. In the latter case, there is no Alexa647 signal but strong GFP signal.
    Figure Legend Snippet: Detecting KCNE2 by 2 antibodies, Ab1 and Ab2. A: transmembrane topology of KCNE2 and a pore-forming α-subunit. B: Ab1 and Ab2 epitopes in the human KCNE2 post-transmembrane domain (TMD) region (yellow shading in A). The human sequence is aligned with those of rat and guinea pig (GP); dot (.) denotes same residue as in the human sequence. Two potential phosphorylation sites in the Ab2 epitope region, Y96 and S98, are marked. C–F: probing KCNE2 expression in young adult (4 to 5 mo) spontaneously hypertensive rat (SHR) ventricular myocytes. SHR myocytes are cultured in serum-free medium for 3 days under the control conditions (control culture) or after overnight incubation with adenovirus carrying green fluorescent protein (Adv-GFP), HA-tagged KCNE2 [Adv-E2 (HA)], or a short hairpin sequence targeting rat KCNE2 nucleotides 319–340 (Adv-E2 si). C–E: immunoblots (IB) of whole cell lysates (WCL) from myocytes in control culture or incubated with adenovirus marked on top. Fresh in E is whole tissue lysate (WTL) prepared from freshly isolated SHR ventricular myocardium. The antibodies used for IB are marked below the images. In C, the central lane is split into 2 and probed with HA mAb (left half) and Ab1 (right half). Size marker positions are marked at left. Solid arrows point to the 2 major HA-tagged KCNE2 bands of 33 and 25 kDa, detected by HA mAb (red arrows; C), Ab1 (black arrows; C), and Ab2 (gray arrows; D). Open arrows point to unrelated proteins detected by HA mAb (C) and by Ab1 or Ab2 (E). Arrows with gradient shade point to the 33-kDa band seen in SHR ventricular myocytes not expressing HA-tagged KCNE2 (C, right, and E). *Highlights the 24-kDa band in WTL from freshly isolated myocardium that is missing in ventricular myocytes after culture under the control conditions (in E). Some lanes are cut and pasted together to avoid showing irrelevant lanes (marked by white stripes between pasted lanes). F: HA immunofluorescence (IF; stained with Alexa647) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence (nuclei) from a myocyte expressing HA-tagged KCNE2 (top) and a myocyte expressing GFP (bottom). In the former case, arrow and arrowhead point to HA IF on cell surface and in perinuclear region. In the latter case, there is no Alexa647 signal but strong GFP signal.

    Techniques Used: Sequencing, Expressing, Cell Culture, Incubation, Western Blot, Isolation, Marker, Immunofluorescence, Staining, Fluorescence

    Ab1 and Ab2 detect similar banding patterns of native KCNE2 proteins in SHR and guinea pig hearts, which are not N-glycosylated. A: PNGase F treatment collapses the ≥20-kDa KCNE2 bands expressed in COS-7 cells into the core unglycosylated 15-kDa band, but does not alter the banding pattern of native proteins in guinea pig and SHR ventricles detected by Ab1. Con, original WTL; +, PNGase F treated; −, similarly processed in the absence of enzyme. B: WTL from guinea pig ventricle probed with Ab2, without or with preincubation of Ab2 with excess Ag (− and +, marked below the image). In both A and B, the solid back arrows point to the 24-kDa native KCNE2 band and arrows with gradient gray point to the 32-kDa putative KCNE2 band. In A, the open gray arrow points to the 15-kDa unglycosylated KCNE2 band. In B, the open black arrow points to a band detected by Ab2 that likely represents an unrelated protein.
    Figure Legend Snippet: Ab1 and Ab2 detect similar banding patterns of native KCNE2 proteins in SHR and guinea pig hearts, which are not N-glycosylated. A: PNGase F treatment collapses the ≥20-kDa KCNE2 bands expressed in COS-7 cells into the core unglycosylated 15-kDa band, but does not alter the banding pattern of native proteins in guinea pig and SHR ventricles detected by Ab1. Con, original WTL; +, PNGase F treated; −, similarly processed in the absence of enzyme. B: WTL from guinea pig ventricle probed with Ab2, without or with preincubation of Ab2 with excess Ag (− and +, marked below the image). In both A and B, the solid back arrows point to the 24-kDa native KCNE2 band and arrows with gradient gray point to the 32-kDa putative KCNE2 band. In A, the open gray arrow points to the 15-kDa unglycosylated KCNE2 band. In B, the open black arrow points to a band detected by Ab2 that likely represents an unrelated protein.

    Techniques Used:

    Quantifying KCNE2 in atria and ventricles of SHR hearts. A: IB of WTL from 4 old (20 mo) SHRs (No. 1-No. 4) with heart-to-body weight ratios (H:B) listed below. After blotting proteins from the gel to a polyvinylidene difluoride (PVDF) membrane, the membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Arrows point to the 2 major 32- and 24-kDa native KCNE2 bands. B: densitometry quantification of KCNE2 protein level. Background-subtracted KCNE2 band intensities are combined, corrected for uneven loading (by dividing the combined KCNE2 band intensities by the actin band intensity of respective lane), and normalized by the mean value of ventricle lanes. **P < 0.01, atria vs. ventricles. C: Ab1 immunofluorescence in SHR ventricular (top) and atrial (bottom) myocytes. Orthogonal dissection of stacks of IF images through the thickness of cells confirms the preferential cell surface KCNE2 localization.
    Figure Legend Snippet: Quantifying KCNE2 in atria and ventricles of SHR hearts. A: IB of WTL from 4 old (20 mo) SHRs (No. 1-No. 4) with heart-to-body weight ratios (H:B) listed below. After blotting proteins from the gel to a polyvinylidene difluoride (PVDF) membrane, the membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Arrows point to the 2 major 32- and 24-kDa native KCNE2 bands. B: densitometry quantification of KCNE2 protein level. Background-subtracted KCNE2 band intensities are combined, corrected for uneven loading (by dividing the combined KCNE2 band intensities by the actin band intensity of respective lane), and normalized by the mean value of ventricle lanes. **P < 0.01, atria vs. ventricles. C: Ab1 immunofluorescence in SHR ventricular (top) and atrial (bottom) myocytes. Orthogonal dissection of stacks of IF images through the thickness of cells confirms the preferential cell surface KCNE2 localization.

    Techniques Used: Immunofluorescence, Dissection

    Quantifying KCNE2 in atria and ventricles of young adult (3 to 4 mo) guinea pig hearts. A: IB of WTL from 4 guinea pigs (No. 1-No. 4). The membrane is probed for KCNE2 with Ab1 (top), stripped, and reprobed for actin. Arrows point to the major 32- and 24-kDa KCNE2 bands. The gray open arrow points to a faint 15-kDa band corresponding to the size of core guinea pig KCNE2 (14.6 kDa). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. *P < 0.05, atria vs. ventricles. C, top: Ab1 IF in freshly isolated guinea pig atrial and ventricular myocytes. C, bottom: HA IF in a cultured guinea pig ventricular myocyte expressing HA-KCNE2 by overnight incubation with Adv-E2 (HA) followed by 24 h culture. Features of HA IF distribution: arrows, striation pattern in cytosol; open triangles, lateral cell surface; open circle, perinuclear; asterisks, punctate.
    Figure Legend Snippet: Quantifying KCNE2 in atria and ventricles of young adult (3 to 4 mo) guinea pig hearts. A: IB of WTL from 4 guinea pigs (No. 1-No. 4). The membrane is probed for KCNE2 with Ab1 (top), stripped, and reprobed for actin. Arrows point to the major 32- and 24-kDa KCNE2 bands. The gray open arrow points to a faint 15-kDa band corresponding to the size of core guinea pig KCNE2 (14.6 kDa). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. *P < 0.05, atria vs. ventricles. C, top: Ab1 IF in freshly isolated guinea pig atrial and ventricular myocytes. C, bottom: HA IF in a cultured guinea pig ventricular myocyte expressing HA-KCNE2 by overnight incubation with Adv-E2 (HA) followed by 24 h culture. Features of HA IF distribution: arrows, striation pattern in cytosol; open triangles, lateral cell surface; open circle, perinuclear; asterisks, punctate.

    Techniques Used: Isolation, Cell Culture, Expressing, Incubation

    Quantifying KCNE2 in 4 human atrial specimens [2 from patients in atrial fibrillation and 2 from patients in sinus rhythm (AF and SR), respectively] and 4 human ventricular specimens [2 from nonfailing and 2 from failing hearts (NF and HF), respectively]. A: membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Coomassie blue (CB) stain confirms even loading (not shown). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. Mean values are shown as histogram bars with SE, and data from individual specimens are shown as symbols.
    Figure Legend Snippet: Quantifying KCNE2 in 4 human atrial specimens [2 from patients in atrial fibrillation and 2 from patients in sinus rhythm (AF and SR), respectively] and 4 human ventricular specimens [2 from nonfailing and 2 from failing hearts (NF and HF), respectively]. A: membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Coomassie blue (CB) stain confirms even loading (not shown). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. Mean values are shown as histogram bars with SE, and data from individual specimens are shown as symbols.

    Techniques Used: Staining

    Detection of KCNE2 by Ab2 requires phosphorylation of serine at position 98 (S98). A: KCNE2 wild-type (WT) and 3 mutants (Y96F, S98A, and Y96F/S98A) are in vitro translated in the presence of γ-32P-ATP to label proteins phosphorylated during translation. The translation products are divided into 2 aliquots, 1 treated with calf intestinal phosphatase (CIP) and the other processed in the same manner without CIP. The aliquots are fractionated by 2 separate SDS-PAGE; 1 is used to probe with Ab1 (top) and the other with Ab2 (middle). Remaining 32P-radioactivity in the gel (in the 10–30 kDa range) is revealed by phosphoimager (bottom). The farthest left lane is no cRNA negative control. B: WCL from COS-7 cells expressing KCNE2 WT or mutants (listed on top), without or with CIP dephosphorylation (− and +, respectively), probed with Ab1 (top) and Ab2 (bottom). C: WTL from dog and human ventricles are divided into 2 aliquots, 1 treated with CIP and the other processed in the same manner without CIP. The 2 aliquots are fractionated side by side and probed with Ab2. CB stain confirms even loading (not shown). In all 3 panels, size marker positions are marked on the right.
    Figure Legend Snippet: Detection of KCNE2 by Ab2 requires phosphorylation of serine at position 98 (S98). A: KCNE2 wild-type (WT) and 3 mutants (Y96F, S98A, and Y96F/S98A) are in vitro translated in the presence of γ-32P-ATP to label proteins phosphorylated during translation. The translation products are divided into 2 aliquots, 1 treated with calf intestinal phosphatase (CIP) and the other processed in the same manner without CIP. The aliquots are fractionated by 2 separate SDS-PAGE; 1 is used to probe with Ab1 (top) and the other with Ab2 (middle). Remaining 32P-radioactivity in the gel (in the 10–30 kDa range) is revealed by phosphoimager (bottom). The farthest left lane is no cRNA negative control. B: WCL from COS-7 cells expressing KCNE2 WT or mutants (listed on top), without or with CIP dephosphorylation (− and +, respectively), probed with Ab1 (top) and Ab2 (bottom). C: WTL from dog and human ventricles are divided into 2 aliquots, 1 treated with CIP and the other processed in the same manner without CIP. The 2 aliquots are fractionated side by side and probed with Ab2. CB stain confirms even loading (not shown). In all 3 panels, size marker positions are marked on the right.

    Techniques Used: In Vitro, SDS Page, Radioactivity, Negative Control, Expressing, De-Phosphorylation Assay, Staining, Marker

    Effects of S98 mutations on KCNE2 modulation of human ether-a-go-go related gene (hERG) current amplitude and protein level. A, a: representative current traces recorded from oocytes injected with cRNA(s) marked on top. A, inset: voltage clamp protocol. A, b: hERG current amplitudes measured from the peak tail currents at −80 mV elicited by 1-s depolarizing pulses to +30 mV and normalized by the mean value from oocytes expressing hERG alone. Data are pooled from 2 independent oocyte expression experiments, with numbers of oocytes studied listed in parentheses. *P < 0.05, vs. hERG alone. B and C: effects of KCNE2 WT, S98A, and S98D on hERG protein levels at the WCL and on the cell surface (biotinylated fraction) in oocytes and in COS-7 cells. Far left lanes are no cRNA or no cDNA negative controls. B and C, top: hERG IB. B and C, bottom: KCNE2 IB using Ab1. CB stain confirms even loading (not shown). hERG migrates as 150- and 300-kDa bands (monomer and dimer). KCNE2 migrates as 15-, 20-, and 25-kDa bands. Each oocyte is injected with 10 ng hERG cRNA and, for coexpression, 6.55 ng KCNE2 cRNA, reaching a cRNA molar ratio (hERG:KCNE2) of 1:5. For COS-7 expression, 2 μg each of hERG and KCNE2 cDNAs are added to 35-mm dish, reaching a cDNA molar ratio (hERG:KCNE2) of 1:1.5.
    Figure Legend Snippet: Effects of S98 mutations on KCNE2 modulation of human ether-a-go-go related gene (hERG) current amplitude and protein level. A, a: representative current traces recorded from oocytes injected with cRNA(s) marked on top. A, inset: voltage clamp protocol. A, b: hERG current amplitudes measured from the peak tail currents at −80 mV elicited by 1-s depolarizing pulses to +30 mV and normalized by the mean value from oocytes expressing hERG alone. Data are pooled from 2 independent oocyte expression experiments, with numbers of oocytes studied listed in parentheses. *P < 0.05, vs. hERG alone. B and C: effects of KCNE2 WT, S98A, and S98D on hERG protein levels at the WCL and on the cell surface (biotinylated fraction) in oocytes and in COS-7 cells. Far left lanes are no cRNA or no cDNA negative controls. B and C, top: hERG IB. B and C, bottom: KCNE2 IB using Ab1. CB stain confirms even loading (not shown). hERG migrates as 150- and 300-kDa bands (monomer and dimer). KCNE2 migrates as 15-, 20-, and 25-kDa bands. Each oocyte is injected with 10 ng hERG cRNA and, for coexpression, 6.55 ng KCNE2 cRNA, reaching a cRNA molar ratio (hERG:KCNE2) of 1:5. For COS-7 expression, 2 μg each of hERG and KCNE2 cDNAs are added to 35-mm dish, reaching a cDNA molar ratio (hERG:KCNE2) of 1:1.5.

    Techniques Used: Injection, Expressing, Staining

    Effect of KCNE2 on hERG turnover rate and the impact of S98A mutation. Details of pulse-chase experiments in COS-7 cells and data interpretation are provided in text. A: quantifying hERG turnover rates when expressed alone or coexpressed with KCNE2. A, top: autoradiographs (35S hERG) and IB of immunoprecipitates from WCL using hERG Ab (IP: hERG) after chase times marked on top. A, bottom: time courses of decay of hERG 35S-radioactivity in immunoprecipitates. Data are normalized by the 0 chase time data point of hERG expressed alone and fit with a single exponential function to estimate the time constants (τ) of hERG turnover (inset, *P < 0.05). B: S98A negates the KCNE2 effect of accelerating hERG turnover. Shown are 35S-autoradiographs of hERG immunoprecipitates from WCL prepared from COS-7 cells expressing the cDNA(s) listed on the left and subjected to pulse-chase experiments using the same procedures as in A. The chase times are listed on top.
    Figure Legend Snippet: Effect of KCNE2 on hERG turnover rate and the impact of S98A mutation. Details of pulse-chase experiments in COS-7 cells and data interpretation are provided in text. A: quantifying hERG turnover rates when expressed alone or coexpressed with KCNE2. A, top: autoradiographs (35S hERG) and IB of immunoprecipitates from WCL using hERG Ab (IP: hERG) after chase times marked on top. A, bottom: time courses of decay of hERG 35S-radioactivity in immunoprecipitates. Data are normalized by the 0 chase time data point of hERG expressed alone and fit with a single exponential function to estimate the time constants (τ) of hERG turnover (inset, *P < 0.05). B: S98A negates the KCNE2 effect of accelerating hERG turnover. Shown are 35S-autoradiographs of hERG immunoprecipitates from WCL prepared from COS-7 cells expressing the cDNA(s) listed on the left and subjected to pulse-chase experiments using the same procedures as in A. The chase times are listed on top.

    Techniques Used: Mutagenesis, Pulse Chase, Radioactivity, Expressing

    Differential effects of KCNE2 on hERG and Kv4.3 protein levels in COS-7 expression. A: KCNE2 reduces whole cell protein level of hERG, but not Kv4.3, in a time-dependent manner. A, top: representative immunoblot images of WCLs, with cDNA(s) transfected and number of posttransfection days listed above. A, bottom: ratios of α-subunit protein level when coexpressed with KCNE2 to when expressed alone, (+KCNE2):(alone), measured on posttransfection day 1. B: pulse-chase experiment in COS-7 cells showing that KCNE2 coexpression does not accelerate Kv4.3 turnover. The format is the same as that of Fig. 8A. As has been reported previously (8), Kv4.3 tends to form high-molecular weight oligomers in addition to the monomer 73-kDa band; the band intensities are combined in quantification. C: Kv4.3 coexpressed with KCNE2 in COS-7 cells is modulated by KCNE2. C, left: KCNE2 slows Kv4.3 activation and inactivation (slowing time to reach peak and decay, currents are recorded at +60 mV). C, middle: KCNE2 shifts the voltage dependence of Kv4.3 activation in the positive direction. C, right: KCNE2 shifts the voltage dependence of Kv4.3 inactivation in the positive direction. Protocols and data analysis have been described previously (31).
    Figure Legend Snippet: Differential effects of KCNE2 on hERG and Kv4.3 protein levels in COS-7 expression. A: KCNE2 reduces whole cell protein level of hERG, but not Kv4.3, in a time-dependent manner. A, top: representative immunoblot images of WCLs, with cDNA(s) transfected and number of posttransfection days listed above. A, bottom: ratios of α-subunit protein level when coexpressed with KCNE2 to when expressed alone, (+KCNE2):(alone), measured on posttransfection day 1. B: pulse-chase experiment in COS-7 cells showing that KCNE2 coexpression does not accelerate Kv4.3 turnover. The format is the same as that of Fig. 8A. As has been reported previously (8), Kv4.3 tends to form high-molecular weight oligomers in addition to the monomer 73-kDa band; the band intensities are combined in quantification. C: Kv4.3 coexpressed with KCNE2 in COS-7 cells is modulated by KCNE2. C, left: KCNE2 slows Kv4.3 activation and inactivation (slowing time to reach peak and decay, currents are recorded at +60 mV). C, middle: KCNE2 shifts the voltage dependence of Kv4.3 activation in the positive direction. C, right: KCNE2 shifts the voltage dependence of Kv4.3 inactivation in the positive direction. Protocols and data analysis have been described previously (31).

    Techniques Used: Expressing, Western Blot, Transfection, Pulse Chase, Molecular Weight, Activation Assay

    Effects of proteasome and lysosome inhibitors [N-acetyl-L-leucyl-L-norleucinal (ALLN) 20 μM and leupeptin 100 μM] on hERG and KCNE2 protein levels in COS-7 WCLs, when the 2 are expressed separately or coexpressed. A: representative immunoblot images of hERG (top) and KCNE2 (with Ab1; middle). The latter is stripped and reprobed for actin (bottom). The cDNA(s) and treatment (control, ALLN, or leupeptin) are marked on top. Size markers are marked on left; hERG- and KCNE2-specific bands are marked on the right (in kDa). B: summary of densitometry quantification. The intensities of background-subtracted hERG- and KCNE2-specific bands are normalized by those of actin bands of respective lanes, and the ratios of ALLN-treated to control (left) or leupeptin-treated to control (right) are calculated for each of the groups. Listed below are transfected cDNA(s) and the numbers of independent COS-7 transfection/immunoblot experiments included in the analysis.
    Figure Legend Snippet: Effects of proteasome and lysosome inhibitors [N-acetyl-L-leucyl-L-norleucinal (ALLN) 20 μM and leupeptin 100 μM] on hERG and KCNE2 protein levels in COS-7 WCLs, when the 2 are expressed separately or coexpressed. A: representative immunoblot images of hERG (top) and KCNE2 (with Ab1; middle). The latter is stripped and reprobed for actin (bottom). The cDNA(s) and treatment (control, ALLN, or leupeptin) are marked on top. Size markers are marked on left; hERG- and KCNE2-specific bands are marked on the right (in kDa). B: summary of densitometry quantification. The intensities of background-subtracted hERG- and KCNE2-specific bands are normalized by those of actin bands of respective lanes, and the ratios of ALLN-treated to control (left) or leupeptin-treated to control (right) are calculated for each of the groups. Listed below are transfected cDNA(s) and the numbers of independent COS-7 transfection/immunoblot experiments included in the analysis.

    Techniques Used: Western Blot, Transfection

    KCNE2 and rapid delayed rectifier K channels (IKr) remodeling during chronic myocardial infarction in canine ventricular myocytes. Chronic myocardial infarction is created by microembolizations as described previously (12). Whole tissue lysates from 4 control and 4 microembolized canine left ventricles are probed with an ERG1a-specific Ab (11) and Ab1 (antibodies listed on the left). The ERG1a-specific Ab recognizes a major 130-kDa dERG1a band, and Ab1 recognizes a major 20-kDa KCNE2 band in canine heart.
    Figure Legend Snippet: KCNE2 and rapid delayed rectifier K channels (IKr) remodeling during chronic myocardial infarction in canine ventricular myocytes. Chronic myocardial infarction is created by microembolizations as described previously (12). Whole tissue lysates from 4 control and 4 microembolized canine left ventricles are probed with an ERG1a-specific Ab (11) and Ab1 (antibodies listed on the left). The ERG1a-specific Ab recognizes a major 130-kDa dERG1a band, and Ab1 recognizes a major 20-kDa KCNE2 band in canine heart.

    Techniques Used:



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    Alomone Labs kcne2 ab1
    Detecting <t>KCNE2</t> by 2 antibodies, <t>Ab1</t> and Ab2. A: transmembrane topology of KCNE2 and a pore-forming α-subunit. B: Ab1 and Ab2 epitopes in the human KCNE2 post-transmembrane domain (TMD) region (yellow shading in A). The human sequence is aligned with those of rat and guinea pig (GP); dot (.) denotes same residue as in the human sequence. Two potential phosphorylation sites in the Ab2 epitope region, Y96 and S98, are marked. C–F: probing KCNE2 expression in young adult (4 to 5 mo) spontaneously hypertensive rat (SHR) ventricular myocytes. SHR myocytes are cultured in serum-free medium for 3 days under the control conditions (control culture) or after overnight incubation with adenovirus carrying green fluorescent protein (Adv-GFP), HA-tagged KCNE2 [Adv-E2 (HA)], or a short hairpin sequence targeting rat KCNE2 nucleotides 319–340 (Adv-E2 si). C–E: immunoblots (IB) of whole cell lysates (WCL) from myocytes in control culture or incubated with adenovirus marked on top. Fresh in E is whole tissue lysate (WTL) prepared from freshly isolated SHR ventricular myocardium. The antibodies used for IB are marked below the images. In C, the central lane is split into 2 and probed with HA mAb (left half) and Ab1 (right half). Size marker positions are marked at left. Solid arrows point to the 2 major HA-tagged KCNE2 bands of 33 and 25 kDa, detected by HA mAb (red arrows; C), Ab1 (black arrows; C), and Ab2 (gray arrows; D). Open arrows point to unrelated proteins detected by HA mAb (C) and by Ab1 or Ab2 (E). Arrows with gradient shade point to the 33-kDa band seen in SHR ventricular myocytes not expressing HA-tagged KCNE2 (C, right, and E). *Highlights the 24-kDa band in WTL from freshly isolated myocardium that is missing in ventricular myocytes after culture under the control conditions (in E). Some lanes are cut and pasted together to avoid showing irrelevant lanes (marked by white stripes between pasted lanes). F: HA immunofluorescence (IF; stained with Alexa647) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence (nuclei) from a myocyte expressing HA-tagged KCNE2 (top) and a myocyte expressing GFP (bottom). In the former case, arrow and arrowhead point to HA IF on cell surface and in perinuclear region. In the latter case, there is no Alexa647 signal but strong GFP signal.
    Kcne2 Ab1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detecting KCNE2 by 2 antibodies, Ab1 and Ab2. A: transmembrane topology of KCNE2 and a pore-forming α-subunit. B: Ab1 and Ab2 epitopes in the human KCNE2 post-transmembrane domain (TMD) region (yellow shading in A). The human sequence is aligned with those of rat and guinea pig (GP); dot (.) denotes same residue as in the human sequence. Two potential phosphorylation sites in the Ab2 epitope region, Y96 and S98, are marked. C–F: probing KCNE2 expression in young adult (4 to 5 mo) spontaneously hypertensive rat (SHR) ventricular myocytes. SHR myocytes are cultured in serum-free medium for 3 days under the control conditions (control culture) or after overnight incubation with adenovirus carrying green fluorescent protein (Adv-GFP), HA-tagged KCNE2 [Adv-E2 (HA)], or a short hairpin sequence targeting rat KCNE2 nucleotides 319–340 (Adv-E2 si). C–E: immunoblots (IB) of whole cell lysates (WCL) from myocytes in control culture or incubated with adenovirus marked on top. Fresh in E is whole tissue lysate (WTL) prepared from freshly isolated SHR ventricular myocardium. The antibodies used for IB are marked below the images. In C, the central lane is split into 2 and probed with HA mAb (left half) and Ab1 (right half). Size marker positions are marked at left. Solid arrows point to the 2 major HA-tagged KCNE2 bands of 33 and 25 kDa, detected by HA mAb (red arrows; C), Ab1 (black arrows; C), and Ab2 (gray arrows; D). Open arrows point to unrelated proteins detected by HA mAb (C) and by Ab1 or Ab2 (E). Arrows with gradient shade point to the 33-kDa band seen in SHR ventricular myocytes not expressing HA-tagged KCNE2 (C, right, and E). *Highlights the 24-kDa band in WTL from freshly isolated myocardium that is missing in ventricular myocytes after culture under the control conditions (in E). Some lanes are cut and pasted together to avoid showing irrelevant lanes (marked by white stripes between pasted lanes). F: HA immunofluorescence (IF; stained with Alexa647) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence (nuclei) from a myocyte expressing HA-tagged KCNE2 (top) and a myocyte expressing GFP (bottom). In the former case, arrow and arrowhead point to HA IF on cell surface and in perinuclear region. In the latter case, there is no Alexa647 signal but strong GFP signal.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Detecting KCNE2 by 2 antibodies, Ab1 and Ab2. A: transmembrane topology of KCNE2 and a pore-forming α-subunit. B: Ab1 and Ab2 epitopes in the human KCNE2 post-transmembrane domain (TMD) region (yellow shading in A). The human sequence is aligned with those of rat and guinea pig (GP); dot (.) denotes same residue as in the human sequence. Two potential phosphorylation sites in the Ab2 epitope region, Y96 and S98, are marked. C–F: probing KCNE2 expression in young adult (4 to 5 mo) spontaneously hypertensive rat (SHR) ventricular myocytes. SHR myocytes are cultured in serum-free medium for 3 days under the control conditions (control culture) or after overnight incubation with adenovirus carrying green fluorescent protein (Adv-GFP), HA-tagged KCNE2 [Adv-E2 (HA)], or a short hairpin sequence targeting rat KCNE2 nucleotides 319–340 (Adv-E2 si). C–E: immunoblots (IB) of whole cell lysates (WCL) from myocytes in control culture or incubated with adenovirus marked on top. Fresh in E is whole tissue lysate (WTL) prepared from freshly isolated SHR ventricular myocardium. The antibodies used for IB are marked below the images. In C, the central lane is split into 2 and probed with HA mAb (left half) and Ab1 (right half). Size marker positions are marked at left. Solid arrows point to the 2 major HA-tagged KCNE2 bands of 33 and 25 kDa, detected by HA mAb (red arrows; C), Ab1 (black arrows; C), and Ab2 (gray arrows; D). Open arrows point to unrelated proteins detected by HA mAb (C) and by Ab1 or Ab2 (E). Arrows with gradient shade point to the 33-kDa band seen in SHR ventricular myocytes not expressing HA-tagged KCNE2 (C, right, and E). *Highlights the 24-kDa band in WTL from freshly isolated myocardium that is missing in ventricular myocytes after culture under the control conditions (in E). Some lanes are cut and pasted together to avoid showing irrelevant lanes (marked by white stripes between pasted lanes). F: HA immunofluorescence (IF; stained with Alexa647) and 4',6-diamidino-2-phenylindole (DAPI) fluorescence (nuclei) from a myocyte expressing HA-tagged KCNE2 (top) and a myocyte expressing GFP (bottom). In the former case, arrow and arrowhead point to HA IF on cell surface and in perinuclear region. In the latter case, there is no Alexa647 signal but strong GFP signal.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Sequencing, Expressing, Cell Culture, Incubation, Western Blot, Isolation, Marker, Immunofluorescence, Staining, Fluorescence

    Ab1 and Ab2 detect similar banding patterns of native KCNE2 proteins in SHR and guinea pig hearts, which are not N-glycosylated. A: PNGase F treatment collapses the ≥20-kDa KCNE2 bands expressed in COS-7 cells into the core unglycosylated 15-kDa band, but does not alter the banding pattern of native proteins in guinea pig and SHR ventricles detected by Ab1. Con, original WTL; +, PNGase F treated; −, similarly processed in the absence of enzyme. B: WTL from guinea pig ventricle probed with Ab2, without or with preincubation of Ab2 with excess Ag (− and +, marked below the image). In both A and B, the solid back arrows point to the 24-kDa native KCNE2 band and arrows with gradient gray point to the 32-kDa putative KCNE2 band. In A, the open gray arrow points to the 15-kDa unglycosylated KCNE2 band. In B, the open black arrow points to a band detected by Ab2 that likely represents an unrelated protein.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Ab1 and Ab2 detect similar banding patterns of native KCNE2 proteins in SHR and guinea pig hearts, which are not N-glycosylated. A: PNGase F treatment collapses the ≥20-kDa KCNE2 bands expressed in COS-7 cells into the core unglycosylated 15-kDa band, but does not alter the banding pattern of native proteins in guinea pig and SHR ventricles detected by Ab1. Con, original WTL; +, PNGase F treated; −, similarly processed in the absence of enzyme. B: WTL from guinea pig ventricle probed with Ab2, without or with preincubation of Ab2 with excess Ag (− and +, marked below the image). In both A and B, the solid back arrows point to the 24-kDa native KCNE2 band and arrows with gradient gray point to the 32-kDa putative KCNE2 band. In A, the open gray arrow points to the 15-kDa unglycosylated KCNE2 band. In B, the open black arrow points to a band detected by Ab2 that likely represents an unrelated protein.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques:

    Quantifying KCNE2 in atria and ventricles of SHR hearts. A: IB of WTL from 4 old (20 mo) SHRs (No. 1-No. 4) with heart-to-body weight ratios (H:B) listed below. After blotting proteins from the gel to a polyvinylidene difluoride (PVDF) membrane, the membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Arrows point to the 2 major 32- and 24-kDa native KCNE2 bands. B: densitometry quantification of KCNE2 protein level. Background-subtracted KCNE2 band intensities are combined, corrected for uneven loading (by dividing the combined KCNE2 band intensities by the actin band intensity of respective lane), and normalized by the mean value of ventricle lanes. **P < 0.01, atria vs. ventricles. C: Ab1 immunofluorescence in SHR ventricular (top) and atrial (bottom) myocytes. Orthogonal dissection of stacks of IF images through the thickness of cells confirms the preferential cell surface KCNE2 localization.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Quantifying KCNE2 in atria and ventricles of SHR hearts. A: IB of WTL from 4 old (20 mo) SHRs (No. 1-No. 4) with heart-to-body weight ratios (H:B) listed below. After blotting proteins from the gel to a polyvinylidene difluoride (PVDF) membrane, the membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Arrows point to the 2 major 32- and 24-kDa native KCNE2 bands. B: densitometry quantification of KCNE2 protein level. Background-subtracted KCNE2 band intensities are combined, corrected for uneven loading (by dividing the combined KCNE2 band intensities by the actin band intensity of respective lane), and normalized by the mean value of ventricle lanes. **P < 0.01, atria vs. ventricles. C: Ab1 immunofluorescence in SHR ventricular (top) and atrial (bottom) myocytes. Orthogonal dissection of stacks of IF images through the thickness of cells confirms the preferential cell surface KCNE2 localization.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Immunofluorescence, Dissection

    Quantifying KCNE2 in atria and ventricles of young adult (3 to 4 mo) guinea pig hearts. A: IB of WTL from 4 guinea pigs (No. 1-No. 4). The membrane is probed for KCNE2 with Ab1 (top), stripped, and reprobed for actin. Arrows point to the major 32- and 24-kDa KCNE2 bands. The gray open arrow points to a faint 15-kDa band corresponding to the size of core guinea pig KCNE2 (14.6 kDa). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. *P < 0.05, atria vs. ventricles. C, top: Ab1 IF in freshly isolated guinea pig atrial and ventricular myocytes. C, bottom: HA IF in a cultured guinea pig ventricular myocyte expressing HA-KCNE2 by overnight incubation with Adv-E2 (HA) followed by 24 h culture. Features of HA IF distribution: arrows, striation pattern in cytosol; open triangles, lateral cell surface; open circle, perinuclear; asterisks, punctate.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Quantifying KCNE2 in atria and ventricles of young adult (3 to 4 mo) guinea pig hearts. A: IB of WTL from 4 guinea pigs (No. 1-No. 4). The membrane is probed for KCNE2 with Ab1 (top), stripped, and reprobed for actin. Arrows point to the major 32- and 24-kDa KCNE2 bands. The gray open arrow points to a faint 15-kDa band corresponding to the size of core guinea pig KCNE2 (14.6 kDa). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. *P < 0.05, atria vs. ventricles. C, top: Ab1 IF in freshly isolated guinea pig atrial and ventricular myocytes. C, bottom: HA IF in a cultured guinea pig ventricular myocyte expressing HA-KCNE2 by overnight incubation with Adv-E2 (HA) followed by 24 h culture. Features of HA IF distribution: arrows, striation pattern in cytosol; open triangles, lateral cell surface; open circle, perinuclear; asterisks, punctate.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Isolation, Cell Culture, Expressing, Incubation

    Quantifying KCNE2 in 4 human atrial specimens [2 from patients in atrial fibrillation and 2 from patients in sinus rhythm (AF and SR), respectively] and 4 human ventricular specimens [2 from nonfailing and 2 from failing hearts (NF and HF), respectively]. A: membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Coomassie blue (CB) stain confirms even loading (not shown). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. Mean values are shown as histogram bars with SE, and data from individual specimens are shown as symbols.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Quantifying KCNE2 in 4 human atrial specimens [2 from patients in atrial fibrillation and 2 from patients in sinus rhythm (AF and SR), respectively] and 4 human ventricular specimens [2 from nonfailing and 2 from failing hearts (NF and HF), respectively]. A: membrane is probed for KCNE2 (Ab1), stripped, and reprobed for actin. Coomassie blue (CB) stain confirms even loading (not shown). B: densitometry quantification of KCNE2 protein level. Data analysis is the same as that described for Fig. 3B. Mean values are shown as histogram bars with SE, and data from individual specimens are shown as symbols.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Staining

    Detection of KCNE2 by Ab2 requires phosphorylation of serine at position 98 (S98). A: KCNE2 wild-type (WT) and 3 mutants (Y96F, S98A, and Y96F/S98A) are in vitro translated in the presence of γ-32P-ATP to label proteins phosphorylated during translation. The translation products are divided into 2 aliquots, 1 treated with calf intestinal phosphatase (CIP) and the other processed in the same manner without CIP. The aliquots are fractionated by 2 separate SDS-PAGE; 1 is used to probe with Ab1 (top) and the other with Ab2 (middle). Remaining 32P-radioactivity in the gel (in the 10–30 kDa range) is revealed by phosphoimager (bottom). The farthest left lane is no cRNA negative control. B: WCL from COS-7 cells expressing KCNE2 WT or mutants (listed on top), without or with CIP dephosphorylation (− and +, respectively), probed with Ab1 (top) and Ab2 (bottom). C: WTL from dog and human ventricles are divided into 2 aliquots, 1 treated with CIP and the other processed in the same manner without CIP. The 2 aliquots are fractionated side by side and probed with Ab2. CB stain confirms even loading (not shown). In all 3 panels, size marker positions are marked on the right.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Detection of KCNE2 by Ab2 requires phosphorylation of serine at position 98 (S98). A: KCNE2 wild-type (WT) and 3 mutants (Y96F, S98A, and Y96F/S98A) are in vitro translated in the presence of γ-32P-ATP to label proteins phosphorylated during translation. The translation products are divided into 2 aliquots, 1 treated with calf intestinal phosphatase (CIP) and the other processed in the same manner without CIP. The aliquots are fractionated by 2 separate SDS-PAGE; 1 is used to probe with Ab1 (top) and the other with Ab2 (middle). Remaining 32P-radioactivity in the gel (in the 10–30 kDa range) is revealed by phosphoimager (bottom). The farthest left lane is no cRNA negative control. B: WCL from COS-7 cells expressing KCNE2 WT or mutants (listed on top), without or with CIP dephosphorylation (− and +, respectively), probed with Ab1 (top) and Ab2 (bottom). C: WTL from dog and human ventricles are divided into 2 aliquots, 1 treated with CIP and the other processed in the same manner without CIP. The 2 aliquots are fractionated side by side and probed with Ab2. CB stain confirms even loading (not shown). In all 3 panels, size marker positions are marked on the right.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: In Vitro, SDS Page, Radioactivity, Negative Control, Expressing, De-Phosphorylation Assay, Staining, Marker

    Effects of S98 mutations on KCNE2 modulation of human ether-a-go-go related gene (hERG) current amplitude and protein level. A, a: representative current traces recorded from oocytes injected with cRNA(s) marked on top. A, inset: voltage clamp protocol. A, b: hERG current amplitudes measured from the peak tail currents at −80 mV elicited by 1-s depolarizing pulses to +30 mV and normalized by the mean value from oocytes expressing hERG alone. Data are pooled from 2 independent oocyte expression experiments, with numbers of oocytes studied listed in parentheses. *P < 0.05, vs. hERG alone. B and C: effects of KCNE2 WT, S98A, and S98D on hERG protein levels at the WCL and on the cell surface (biotinylated fraction) in oocytes and in COS-7 cells. Far left lanes are no cRNA or no cDNA negative controls. B and C, top: hERG IB. B and C, bottom: KCNE2 IB using Ab1. CB stain confirms even loading (not shown). hERG migrates as 150- and 300-kDa bands (monomer and dimer). KCNE2 migrates as 15-, 20-, and 25-kDa bands. Each oocyte is injected with 10 ng hERG cRNA and, for coexpression, 6.55 ng KCNE2 cRNA, reaching a cRNA molar ratio (hERG:KCNE2) of 1:5. For COS-7 expression, 2 μg each of hERG and KCNE2 cDNAs are added to 35-mm dish, reaching a cDNA molar ratio (hERG:KCNE2) of 1:1.5.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Effects of S98 mutations on KCNE2 modulation of human ether-a-go-go related gene (hERG) current amplitude and protein level. A, a: representative current traces recorded from oocytes injected with cRNA(s) marked on top. A, inset: voltage clamp protocol. A, b: hERG current amplitudes measured from the peak tail currents at −80 mV elicited by 1-s depolarizing pulses to +30 mV and normalized by the mean value from oocytes expressing hERG alone. Data are pooled from 2 independent oocyte expression experiments, with numbers of oocytes studied listed in parentheses. *P < 0.05, vs. hERG alone. B and C: effects of KCNE2 WT, S98A, and S98D on hERG protein levels at the WCL and on the cell surface (biotinylated fraction) in oocytes and in COS-7 cells. Far left lanes are no cRNA or no cDNA negative controls. B and C, top: hERG IB. B and C, bottom: KCNE2 IB using Ab1. CB stain confirms even loading (not shown). hERG migrates as 150- and 300-kDa bands (monomer and dimer). KCNE2 migrates as 15-, 20-, and 25-kDa bands. Each oocyte is injected with 10 ng hERG cRNA and, for coexpression, 6.55 ng KCNE2 cRNA, reaching a cRNA molar ratio (hERG:KCNE2) of 1:5. For COS-7 expression, 2 μg each of hERG and KCNE2 cDNAs are added to 35-mm dish, reaching a cDNA molar ratio (hERG:KCNE2) of 1:1.5.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Injection, Expressing, Staining

    Effect of KCNE2 on hERG turnover rate and the impact of S98A mutation. Details of pulse-chase experiments in COS-7 cells and data interpretation are provided in text. A: quantifying hERG turnover rates when expressed alone or coexpressed with KCNE2. A, top: autoradiographs (35S hERG) and IB of immunoprecipitates from WCL using hERG Ab (IP: hERG) after chase times marked on top. A, bottom: time courses of decay of hERG 35S-radioactivity in immunoprecipitates. Data are normalized by the 0 chase time data point of hERG expressed alone and fit with a single exponential function to estimate the time constants (τ) of hERG turnover (inset, *P < 0.05). B: S98A negates the KCNE2 effect of accelerating hERG turnover. Shown are 35S-autoradiographs of hERG immunoprecipitates from WCL prepared from COS-7 cells expressing the cDNA(s) listed on the left and subjected to pulse-chase experiments using the same procedures as in A. The chase times are listed on top.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Effect of KCNE2 on hERG turnover rate and the impact of S98A mutation. Details of pulse-chase experiments in COS-7 cells and data interpretation are provided in text. A: quantifying hERG turnover rates when expressed alone or coexpressed with KCNE2. A, top: autoradiographs (35S hERG) and IB of immunoprecipitates from WCL using hERG Ab (IP: hERG) after chase times marked on top. A, bottom: time courses of decay of hERG 35S-radioactivity in immunoprecipitates. Data are normalized by the 0 chase time data point of hERG expressed alone and fit with a single exponential function to estimate the time constants (τ) of hERG turnover (inset, *P < 0.05). B: S98A negates the KCNE2 effect of accelerating hERG turnover. Shown are 35S-autoradiographs of hERG immunoprecipitates from WCL prepared from COS-7 cells expressing the cDNA(s) listed on the left and subjected to pulse-chase experiments using the same procedures as in A. The chase times are listed on top.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Mutagenesis, Pulse Chase, Radioactivity, Expressing

    Differential effects of KCNE2 on hERG and Kv4.3 protein levels in COS-7 expression. A: KCNE2 reduces whole cell protein level of hERG, but not Kv4.3, in a time-dependent manner. A, top: representative immunoblot images of WCLs, with cDNA(s) transfected and number of posttransfection days listed above. A, bottom: ratios of α-subunit protein level when coexpressed with KCNE2 to when expressed alone, (+KCNE2):(alone), measured on posttransfection day 1. B: pulse-chase experiment in COS-7 cells showing that KCNE2 coexpression does not accelerate Kv4.3 turnover. The format is the same as that of Fig. 8A. As has been reported previously (8), Kv4.3 tends to form high-molecular weight oligomers in addition to the monomer 73-kDa band; the band intensities are combined in quantification. C: Kv4.3 coexpressed with KCNE2 in COS-7 cells is modulated by KCNE2. C, left: KCNE2 slows Kv4.3 activation and inactivation (slowing time to reach peak and decay, currents are recorded at +60 mV). C, middle: KCNE2 shifts the voltage dependence of Kv4.3 activation in the positive direction. C, right: KCNE2 shifts the voltage dependence of Kv4.3 inactivation in the positive direction. Protocols and data analysis have been described previously (31).

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Differential effects of KCNE2 on hERG and Kv4.3 protein levels in COS-7 expression. A: KCNE2 reduces whole cell protein level of hERG, but not Kv4.3, in a time-dependent manner. A, top: representative immunoblot images of WCLs, with cDNA(s) transfected and number of posttransfection days listed above. A, bottom: ratios of α-subunit protein level when coexpressed with KCNE2 to when expressed alone, (+KCNE2):(alone), measured on posttransfection day 1. B: pulse-chase experiment in COS-7 cells showing that KCNE2 coexpression does not accelerate Kv4.3 turnover. The format is the same as that of Fig. 8A. As has been reported previously (8), Kv4.3 tends to form high-molecular weight oligomers in addition to the monomer 73-kDa band; the band intensities are combined in quantification. C: Kv4.3 coexpressed with KCNE2 in COS-7 cells is modulated by KCNE2. C, left: KCNE2 slows Kv4.3 activation and inactivation (slowing time to reach peak and decay, currents are recorded at +60 mV). C, middle: KCNE2 shifts the voltage dependence of Kv4.3 activation in the positive direction. C, right: KCNE2 shifts the voltage dependence of Kv4.3 inactivation in the positive direction. Protocols and data analysis have been described previously (31).

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Expressing, Western Blot, Transfection, Pulse Chase, Molecular Weight, Activation Assay

    Effects of proteasome and lysosome inhibitors [N-acetyl-L-leucyl-L-norleucinal (ALLN) 20 μM and leupeptin 100 μM] on hERG and KCNE2 protein levels in COS-7 WCLs, when the 2 are expressed separately or coexpressed. A: representative immunoblot images of hERG (top) and KCNE2 (with Ab1; middle). The latter is stripped and reprobed for actin (bottom). The cDNA(s) and treatment (control, ALLN, or leupeptin) are marked on top. Size markers are marked on left; hERG- and KCNE2-specific bands are marked on the right (in kDa). B: summary of densitometry quantification. The intensities of background-subtracted hERG- and KCNE2-specific bands are normalized by those of actin bands of respective lanes, and the ratios of ALLN-treated to control (left) or leupeptin-treated to control (right) are calculated for each of the groups. Listed below are transfected cDNA(s) and the numbers of independent COS-7 transfection/immunoblot experiments included in the analysis.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: Effects of proteasome and lysosome inhibitors [N-acetyl-L-leucyl-L-norleucinal (ALLN) 20 μM and leupeptin 100 μM] on hERG and KCNE2 protein levels in COS-7 WCLs, when the 2 are expressed separately or coexpressed. A: representative immunoblot images of hERG (top) and KCNE2 (with Ab1; middle). The latter is stripped and reprobed for actin (bottom). The cDNA(s) and treatment (control, ALLN, or leupeptin) are marked on top. Size markers are marked on left; hERG- and KCNE2-specific bands are marked on the right (in kDa). B: summary of densitometry quantification. The intensities of background-subtracted hERG- and KCNE2-specific bands are normalized by those of actin bands of respective lanes, and the ratios of ALLN-treated to control (left) or leupeptin-treated to control (right) are calculated for each of the groups. Listed below are transfected cDNA(s) and the numbers of independent COS-7 transfection/immunoblot experiments included in the analysis.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: Western Blot, Transfection

    KCNE2 and rapid delayed rectifier K channels (IKr) remodeling during chronic myocardial infarction in canine ventricular myocytes. Chronic myocardial infarction is created by microembolizations as described previously (12). Whole tissue lysates from 4 control and 4 microembolized canine left ventricles are probed with an ERG1a-specific Ab (11) and Ab1 (antibodies listed on the left). The ERG1a-specific Ab recognizes a major 130-kDa dERG1a band, and Ab1 recognizes a major 20-kDa KCNE2 band in canine heart.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

    doi: 10.1152/ajpheart.00691.2011

    Figure Lengend Snippet: KCNE2 and rapid delayed rectifier K channels (IKr) remodeling during chronic myocardial infarction in canine ventricular myocytes. Chronic myocardial infarction is created by microembolizations as described previously (12). Whole tissue lysates from 4 control and 4 microembolized canine left ventricles are probed with an ERG1a-specific Ab (11) and Ab1 (antibodies listed on the left). The ERG1a-specific Ab recognizes a major 130-kDa dERG1a band, and Ab1 recognizes a major 20-kDa KCNE2 band in canine heart.

    Article Snippet: The following antibodies are used: KCNE2 Ab1 (made by ourselves) and Ab2 (Alomone), HA mAb (Covance), actin mAb (Sigma), hERG pAb (Alomone), ERG1a pAb (a kind gift from Dr. Gail A. Robertson, University of Wisconsin at Madison) ( 11 ), and Kv4.3 mAb (NeuroMab).

    Techniques: