Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Molecular expression levels of TMEM16A in LV. (A) TMEM16A mRNA expression level in LV ( n = 3 mice in each group per time point, RM two-way ANOVA). (B) Traditional western blot results of TMEM16A protein expression level in LV ( n = 3 mice in each group per time point, RM two-way ANOVA). (C) Simple western blot results of TMEM16A protein expression level in LV ( n = 3 mice in each group per time point, RM two-way ANOVA).

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: I to in LVMs. (A) Recordings show the effects of T16A inh -A01 (30 μM) or 4-AP (5 mM) on I to in LVMs. (B) Recordings show the effects of Anti-TMEM16A antibody (1:200) or 4-AP (5 mM) on I to in LVMs. (C) I-V relations of T16A inh -A01 sensitive current and Anti-TMEM16A antibody sensitive current ( n = 4 cells in each group, two-way ANOVA). (D) I-V relation of 4-AP sensitive current ( n = 4 cells in each group, two-way ANOVA).

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques:

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: I TMEM16A in LVMs. (A) Superimposed current tracings were recorded from the same cell before (control) and after exposure to 30 μM T16A inh -A01. Right panel shows expanded traces in the blue box of left panel. (B) Bar graphs show average T16A inh -A01 sensitive current densities in LVMs ( n = 7—8 cells from three mice in each group per time point, RM two-way ANOVA).

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques:

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Molecular expression levels of TMEM16A in HUVECs. (A) TMEM16A mRNA expression level in HUVECs (n = 3–4). (B) Western blot results of TMEM16A protein expression level in HUVECs ( n = 3–5). * p < 0.05, ** p < 0.01 and *** p < 0.001, one-way ANOVA and unpaired t -test.

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Effect of TMEM16A on migration in HUVECs. (A) Wound healing assay results of HUVECs transfected with siRNA NC or siRNA Mix , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately ( n = 5—8 in each group per time point). (B) Wound healing assay results of HUVECs transfected with TM NC or TM OE , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately (n = 5 – 7 in each group per time point). * p < 0.05 and ** p < 0.01 compared with siRNA NC or TM NC under the same treatment and at the same time point, # p < 0.05 and ## p < 0.01 compared with vehicle (PBS) in the same cell type and at the same time point, RM two-way ANOVA.

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Migration, Wound Healing Assay, Transfection

Journal: Frontiers in Physiology

Article Title: TMEM16A Plays an Insignificant Role in Myocardium Remodeling but May Promote Angiogenesis of Heart During Pressure-overload

doi: 10.3389/fphys.2022.897619

Figure Lengend Snippet: Effect of TMEM16A on angiogenesis in HUVECs. (A,C) Tube formation assay results of HUVECs transfected with siRNA NC or siRNA Mix , or TM NC , or TM OE , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately (n = 3–9). (B,D) Endothelial cell spheroids sprouting assay results of HUVECs transfected with siRNA NC or siRNA Mix , or TM NC , or TM OE , and treated with vehicle (PBS), or VEGF (30 ng/mL), or AngII (1 μM) separately ( n = 3–5). * p < 0.05 compared with siRNA NC or TM NC under the same treatment, # p < 0.05 and ## p < 0.01 compared with vehicle (PBS) in the same cell type, two-way ANOVA.

Article Snippet: Briefly, total protein was separated on 10% SDS-PAGE, transferred onto a PVDF membrane and blotted with the following primary antibodies separately: Anti-TMEM16A antibody (ACL-011, Alomone labs) for protein extracted from mouse heart, Anti-TMEM16A antibody (BA3464-2, Boster) for endogenous human TMEM16A protein in HUVECs, Anti-TMEM16A antibody (ab53212, Abcam) for overexpressed mouse TMEM16A protein induced by lentivirus in HUVECs, and Anti-β-actin antibody (CST).

Techniques: Tube Formation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: PK cleaves Kv1.5 at the S1–S2 linker and increases IKv1.5. A, Western blots depicting Kv1.5 expression following PK treatment (200 μg/ml, 20 min, 37 °C) of Kv1.5-HEK cells cultured with or without Tuni (10 μg/ml) for 48 h (n = 6). Proteins were detected using anti-N-terminal (N-Ab) or anti-C-terminal (C-Ab) Kv1.5 antibody. Actin was used as a loading control. Molecular mass marker (Marker) is shown in the middle. B, schematic illustration of Kv1.5 PK cleavage. C, co-IP assay showing that the N- (N-FR) and C-fragments (C-FR) do not associate after PK cleavage. Whole-cell proteins were extracted from PK-treated WT Kv1.5-HEK cells, and an anti-N-terminal Kv1.5 antibody was added to precipitate the N-fragment and associated proteins. Although the N-fragment was detected in the precipitate, the C-fragment was not detected. Western blotting (WB) of uncleaved and PK-cleaved Kv1.5 is shown to indicate the fragments. GAPDH was used as the control. IP, immunoprecipitation; IB, immunoblotting. The same results were obtained from five co-IP experiments. D, IKv1.5 in control (CTL) and PK-treated cells. The voltage protocol is shown above the current traces, and the summarized current-voltage (I-V) and g-V relationships are shown beneath the current traces (n = 36 in control; n = 35 in PK cleavage; **, p < 0.01 at 0 mV and above). Error bars represent S.E.

Article Snippet: An S1–S2 linker–specific anti-Kv1.5 antibody (APC-150) raised against amino acid residues 268–279 of rat Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"P19024","term_id":"116433","term_text":"P19024"}} P19024 ; 11 of 12 amino acid residues identical to human Kv1.5 amino acids 277–288) was purchased from Alomone Labs. MEM, fetal bovine serum, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, Opti-MEM, Oregon Green 488 wheat germ agglutinin, Hanks' balanced salt solution, and Alexa Fluor 594–conjugated donkey anti-rabbit secondary antibody were purchased from Invitrogen.

Techniques: Western Blot, Expressing, Cell Culture, Marker, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Expression and function of Kv1.5 N- (N-FR) and C-fragments (C-FR). A and B, Western blots of Frag(1–303) and Frag(304–613) along with full-length Kv1.5 protein without and with PK cleavage from cells treated without or with Tuni treatment (10 μg/ml, 48 h). Kv1.5 proteins were detected with an N-terminal (N-Ab) or C-terminal antibody (C-Ab). Actin was used as a loading control (n = 8). C–E, schematics illustrating Frag(1–303) and Frag(304–613) as well as corresponding current traces upon independent expression or coexpression. The voltage protocol is shown in the inset above the current traces. F, summarized current-voltage relationships of currents generated by Frag(1–303) (n = 8), Frag(304–613) (n = 10), and Frag(1–303)+(304–613) (n = 14). Error bars represent S.E. CTL, control.

Article Snippet: An S1–S2 linker–specific anti-Kv1.5 antibody (APC-150) raised against amino acid residues 268–279 of rat Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"P19024","term_id":"116433","term_text":"P19024"}} P19024 ; 11 of 12 amino acid residues identical to human Kv1.5 amino acids 277–288) was purchased from Alomone Labs. MEM, fetal bovine serum, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, Opti-MEM, Oregon Green 488 wheat germ agglutinin, Hanks' balanced salt solution, and Alexa Fluor 594–conjugated donkey anti-rabbit secondary antibody were purchased from Invitrogen.

Techniques: Expressing, Western Blot, Generated

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Frag(304–613) requires Frag(1–303) to traffic to the plasma membrane. A, Western blots depicting total expression (left) and membrane expression (right) of Frag(1–303) and Frag(304–613) expressed independently or together (n = 6). Actin and Na+/K+-ATPase were used as loading controls for total and membrane proteins, respectively. Molecular mass marker (Marker) is shown in the middle. B, confocal images portraying the localization of WT Kv1.5, Frag(1–303), or Frag(304–613) relative to the plasma membrane (n = 4). WT Kv1.5 was detected with the N-terminal antibody and is depicted in red. Frag(1–303) and Frag(304–613), detected with N-terminal (N-Ab) and C-terminal (C-Ab) antibodies, respectively, are depicted in red. Cell membranes are shown in green. Fluorescence intensities of WT Kv1.5, Frag(1–303), and/or Frag(304–613) and the membrane in the line across the cells were quantified by ImageJ and are shown beneath the representative images.

Article Snippet: An S1–S2 linker–specific anti-Kv1.5 antibody (APC-150) raised against amino acid residues 268–279 of rat Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"P19024","term_id":"116433","term_text":"P19024"}} P19024 ; 11 of 12 amino acid residues identical to human Kv1.5 amino acids 277–288) was purchased from Alomone Labs. MEM, fetal bovine serum, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, Opti-MEM, Oregon Green 488 wheat germ agglutinin, Hanks' balanced salt solution, and Alexa Fluor 594–conjugated donkey anti-rabbit secondary antibody were purchased from Invitrogen.

Techniques: Western Blot, Expressing, Marker, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Dominant-negative suppression of WT Kv1.5 with Frag(1–303). Shown are the effects of transfecting empty pcDNA3 (PC3; control; n = 27) or plasmids encoding Frag(1–209) (n = 12), Frag(210–303) (n = 11), or Frag(1–303) (n = 17) into Kv1.5-HEK cells on IKv1.5. Representative current traces are depicted above the summarized box plots of current amplitudes upon 50-mV depolarization. **, p < 0.01 versus empty pcDNA3.

Article Snippet: An S1–S2 linker–specific anti-Kv1.5 antibody (APC-150) raised against amino acid residues 268–279 of rat Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"P19024","term_id":"116433","term_text":"P19024"}} P19024 ; 11 of 12 amino acid residues identical to human Kv1.5 amino acids 277–288) was purchased from Alomone Labs. MEM, fetal bovine serum, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, Opti-MEM, Oregon Green 488 wheat germ agglutinin, Hanks' balanced salt solution, and Alexa Fluor 594–conjugated donkey anti-rabbit secondary antibody were purchased from Invitrogen.

Techniques: Dominant Negative Mutation

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: The T1–S1 linker and/or S1 plays a role in Kv1.5 membrane insertion. A, Western blots of WT, ΔN209, and del(2–240) Kv1.5 channels expressed in HEK cells (n = 5). Molecular mass marker (Marker) is shown on the right. B, schematic illustration of WT, ΔN209, and del(2–240) Kv1.5 channels along with their representative current traces. The voltage protocol was the same as in Fig. 2. On the right are summarized I-V relationships of WT (n = 12), ΔN209 (n = 6), and del(2–240) Kv1.5 channels (n = 8). **, p < 0.01 at −10 mV and above between ΔN209 and WT Kv1.5 currents. C, Western blot of del(2–240) Kv1.5 protein from transfected cells cultured in standard condition (CTL), with Tuni treatment (10 μg/ml, 48 h), with PK treatment (200 μg/ml, 20 min, 37 °C), or with BFA treatment (10 μm, 12 h). Actin was used as a loading control. Summarized bar graphs are depicted below the representative Western blot images (n = 4). D, confocal images portraying the cellular location of ΔN209 or del(2–240) Kv1.5 proteins relative to the plasma membrane (n = 5). ΔN209 or del(2–240) Kv1.5 proteins were stained with a C-terminal antibody (C-Ab) (red). Cell membranes are stained green. Fluorescence intensities of ΔN209 or del(2–240) Kv1.5 (red) and the membrane (green) in the line across the cell were quantified by ImageJ and are shown on the right. Error bars represent S.E.

Article Snippet: An S1–S2 linker–specific anti-Kv1.5 antibody (APC-150) raised against amino acid residues 268–279 of rat Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"P19024","term_id":"116433","term_text":"P19024"}} P19024 ; 11 of 12 amino acid residues identical to human Kv1.5 amino acids 277–288) was purchased from Alomone Labs. MEM, fetal bovine serum, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, Opti-MEM, Oregon Green 488 wheat germ agglutinin, Hanks' balanced salt solution, and Alexa Fluor 594–conjugated donkey anti-rabbit secondary antibody were purchased from Invitrogen.

Techniques: Western Blot, Marker, Transfection, Cell Culture, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Voltage-dependent inactivation of WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels. A, left, schematic illustration of the channels. Middle, representative current traces recorded using the voltage protocol shown above from cells expressing the channels illustrated. The interpulse interval was 15 s. Right, voltage dependence of inactivation. Currents at 60 mV were normalized to the maximum value and plotted versus the test voltages for WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels (n = 7–11 cells from three independent experiments for each). B, excessive cumulative inactivation of ΔN209 and Frag(1–303)+(304–613)-coassembled Kv1.5 channels compared with WT Kv1.5. The voltage protocol is depicted above plots of normalized current amplitudes against depolarizing steps relative to the value upon the first step (n = 6–8 cells for each channel). Error bars represent S.E.

Article Snippet: An S1–S2 linker–specific anti-Kv1.5 antibody (APC-150) raised against amino acid residues 268–279 of rat Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"P19024","term_id":"116433","term_text":"P19024"}} P19024 ; 11 of 12 amino acid residues identical to human Kv1.5 amino acids 277–288) was purchased from Alomone Labs. MEM, fetal bovine serum, trypsin, sodium pyruvate, minimal essential amino acids, Lipofectamine 2000, Opti-MEM, Oregon Green 488 wheat germ agglutinin, Hanks' balanced salt solution, and Alexa Fluor 594–conjugated donkey anti-rabbit secondary antibody were purchased from Invitrogen.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: PK cleaves Kv1.5 at the S1–S2 linker and increases IKv1.5. A, Western blots depicting Kv1.5 expression following PK treatment (200 μg/ml, 20 min, 37 °C) of Kv1.5-HEK cells cultured with or without Tuni (10 μg/ml) for 48 h (n = 6). Proteins were detected using anti-N-terminal (N-Ab) or anti-C-terminal (C-Ab) Kv1.5 antibody. Actin was used as a loading control. Molecular mass marker (Marker) is shown in the middle. B, schematic illustration of Kv1.5 PK cleavage. C, co-IP assay showing that the N- (N-FR) and C-fragments (C-FR) do not associate after PK cleavage. Whole-cell proteins were extracted from PK-treated WT Kv1.5-HEK cells, and an anti-N-terminal Kv1.5 antibody was added to precipitate the N-fragment and associated proteins. Although the N-fragment was detected in the precipitate, the C-fragment was not detected. Western blotting (WB) of uncleaved and PK-cleaved Kv1.5 is shown to indicate the fragments. GAPDH was used as the control. IP, immunoprecipitation; IB, immunoblotting. The same results were obtained from five co-IP experiments. D, IKv1.5 in control (CTL) and PK-treated cells. The voltage protocol is shown above the current traces, and the summarized current-voltage (I-V) and g-V relationships are shown beneath the current traces (n = 36 in control; n = 35 in PK cleavage; **, p < 0.01 at 0 mV and above). Error bars represent S.E.

Article Snippet: A C terminus–specific anti-Kv1.5 antibody (APC-004) raised against amino acid residues 513–602 of mouse Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"Q61762","term_id":"341940859","term_text":"Q61762"}} Q61762 ; 70 of 90 amino acid residues identical to human Kv1.5 amino acids 524–613) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Expressing, Cell Culture, Marker, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Expression and function of Kv1.5 N- (N-FR) and C-fragments (C-FR). A and B, Western blots of Frag(1–303) and Frag(304–613) along with full-length Kv1.5 protein without and with PK cleavage from cells treated without or with Tuni treatment (10 μg/ml, 48 h). Kv1.5 proteins were detected with an N-terminal (N-Ab) or C-terminal antibody (C-Ab). Actin was used as a loading control (n = 8). C–E, schematics illustrating Frag(1–303) and Frag(304–613) as well as corresponding current traces upon independent expression or coexpression. The voltage protocol is shown in the inset above the current traces. F, summarized current-voltage relationships of currents generated by Frag(1–303) (n = 8), Frag(304–613) (n = 10), and Frag(1–303)+(304–613) (n = 14). Error bars represent S.E. CTL, control.

Article Snippet: A C terminus–specific anti-Kv1.5 antibody (APC-004) raised against amino acid residues 513–602 of mouse Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"Q61762","term_id":"341940859","term_text":"Q61762"}} Q61762 ; 70 of 90 amino acid residues identical to human Kv1.5 amino acids 524–613) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Expressing, Western Blot, Generated

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Frag(304–613) requires Frag(1–303) to traffic to the plasma membrane. A, Western blots depicting total expression (left) and membrane expression (right) of Frag(1–303) and Frag(304–613) expressed independently or together (n = 6). Actin and Na+/K+-ATPase were used as loading controls for total and membrane proteins, respectively. Molecular mass marker (Marker) is shown in the middle. B, confocal images portraying the localization of WT Kv1.5, Frag(1–303), or Frag(304–613) relative to the plasma membrane (n = 4). WT Kv1.5 was detected with the N-terminal antibody and is depicted in red. Frag(1–303) and Frag(304–613), detected with N-terminal (N-Ab) and C-terminal (C-Ab) antibodies, respectively, are depicted in red. Cell membranes are shown in green. Fluorescence intensities of WT Kv1.5, Frag(1–303), and/or Frag(304–613) and the membrane in the line across the cells were quantified by ImageJ and are shown beneath the representative images.

Article Snippet: A C terminus–specific anti-Kv1.5 antibody (APC-004) raised against amino acid residues 513–602 of mouse Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"Q61762","term_id":"341940859","term_text":"Q61762"}} Q61762 ; 70 of 90 amino acid residues identical to human Kv1.5 amino acids 524–613) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Expressing, Marker, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Dominant-negative suppression of WT Kv1.5 with Frag(1–303). Shown are the effects of transfecting empty pcDNA3 (PC3; control; n = 27) or plasmids encoding Frag(1–209) (n = 12), Frag(210–303) (n = 11), or Frag(1–303) (n = 17) into Kv1.5-HEK cells on IKv1.5. Representative current traces are depicted above the summarized box plots of current amplitudes upon 50-mV depolarization. **, p < 0.01 versus empty pcDNA3.

Article Snippet: A C terminus–specific anti-Kv1.5 antibody (APC-004) raised against amino acid residues 513–602 of mouse Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"Q61762","term_id":"341940859","term_text":"Q61762"}} Q61762 ; 70 of 90 amino acid residues identical to human Kv1.5 amino acids 524–613) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Dominant Negative Mutation

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: The T1–S1 linker and/or S1 plays a role in Kv1.5 membrane insertion. A, Western blots of WT, ΔN209, and del(2–240) Kv1.5 channels expressed in HEK cells (n = 5). Molecular mass marker (Marker) is shown on the right. B, schematic illustration of WT, ΔN209, and del(2–240) Kv1.5 channels along with their representative current traces. The voltage protocol was the same as in Fig. 2. On the right are summarized I-V relationships of WT (n = 12), ΔN209 (n = 6), and del(2–240) Kv1.5 channels (n = 8). **, p < 0.01 at −10 mV and above between ΔN209 and WT Kv1.5 currents. C, Western blot of del(2–240) Kv1.5 protein from transfected cells cultured in standard condition (CTL), with Tuni treatment (10 μg/ml, 48 h), with PK treatment (200 μg/ml, 20 min, 37 °C), or with BFA treatment (10 μm, 12 h). Actin was used as a loading control. Summarized bar graphs are depicted below the representative Western blot images (n = 4). D, confocal images portraying the cellular location of ΔN209 or del(2–240) Kv1.5 proteins relative to the plasma membrane (n = 5). ΔN209 or del(2–240) Kv1.5 proteins were stained with a C-terminal antibody (C-Ab) (red). Cell membranes are stained green. Fluorescence intensities of ΔN209 or del(2–240) Kv1.5 (red) and the membrane (green) in the line across the cell were quantified by ImageJ and are shown on the right. Error bars represent S.E.

Article Snippet: A C terminus–specific anti-Kv1.5 antibody (APC-004) raised against amino acid residues 513–602 of mouse Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"Q61762","term_id":"341940859","term_text":"Q61762"}} Q61762 ; 70 of 90 amino acid residues identical to human Kv1.5 amino acids 524–613) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Western Blot, Marker, Transfection, Cell Culture, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

doi: 10.1074/jbc.RA118.004065

Figure Lengend Snippet: Voltage-dependent inactivation of WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels. A, left, schematic illustration of the channels. Middle, representative current traces recorded using the voltage protocol shown above from cells expressing the channels illustrated. The interpulse interval was 15 s. Right, voltage dependence of inactivation. Currents at 60 mV were normalized to the maximum value and plotted versus the test voltages for WT, ΔN209, and Frag(1–303)+(304–613)-coassembled Kv1.5 channels (n = 7–11 cells from three independent experiments for each). B, excessive cumulative inactivation of ΔN209 and Frag(1–303)+(304–613)-coassembled Kv1.5 channels compared with WT Kv1.5. The voltage protocol is depicted above plots of normalized current amplitudes against depolarizing steps relative to the value upon the first step (n = 6–8 cells for each channel). Error bars represent S.E.

Article Snippet: A C terminus–specific anti-Kv1.5 antibody (APC-004) raised against amino acid residues 513–602 of mouse Kv1.5 (accession number {"type":"entrez-protein","attrs":{"text":"Q61762","term_id":"341940859","term_text":"Q61762"}} Q61762 ; 70 of 90 amino acid residues identical to human Kv1.5 amino acids 524–613) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Expressing