Journal: PLoS ONE

Article Title: Effect of Methamphetamine on the Microglial Damage: Role of Potassium Channel Kv1.3

doi: 10.1371/journal.pone.0088642

Figure Lengend Snippet: (A) Meth significantly increased the Kv1.3 mRNA expression, while MgTx ameliorated the expression of Kv1.3 mediated by Meth. (B) Meth showed no obvious impact on Kv1.5 mRNA expression. Each data point represents mean±SD of mRNA levels from at least three separate experiments in which treatments were performed in triplicates.* and # indicate significant differences compared with the control and condition stimulated by Meth respectively (p<0.05 by ANOVA), each data represents mean±SD at least three separate experiments.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the polyclonal Kv1.3, Kv1.5 antibody (Alomone Labs,Israel).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Effect of Methamphetamine on the Microglial Damage: Role of Potassium Channel Kv1.3

doi: 10.1371/journal.pone.0088642

Figure Lengend Snippet: Microglial cells were incubated with Meth, and the level of Kv1.3, Kv1.5 proteins were detected by western-blot. (A) Meth (0, 100,300 µM) up-regulated Kv1.3 protein in a concentration dependent manner. (B) Meth (300 µM) obviously up-regulated Kv1.3 protein expression in 12, 24 and 48 h, and displayed a time-dependent way. (C) Meth mediated up-regulation of Kv1.3 protein level was partly attenuated by MgTx (10 nM). (D) and (E) Meth (300 µM) showed no impact on Kv1.5 protein level. * and # indicate significant differences compared with the control and condition stimulated by Meth respectively (p<0.05 by ANOVA), each data represents mean±SD at least three separate experiments.

Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with the polyclonal Kv1.3, Kv1.5 antibody (Alomone Labs,Israel).

Techniques: Incubation, Western Blot, Concentration Assay, Expressing

Journal: Reproductive biology and endocrinology : RB&E

Article Title: The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

doi: 10.1186/1477-7827-5-41

Figure Lengend Snippet: Kv channel α subunit proteins are expressed in mouse myometrium . Results obtained from Western blots demonstrating the presence of immunoreactive bands for Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv1.6, and Kv4.2 α-subunits in myometrium from nonpregnant (NP) and term-pregnant (P20) mice. Kv4.3 α-subunits were detected in the NP samples, but were undetectable in the term-pregnant mouse myometrium. Apparent molecular weights of each protein species are indicated at right. Different lanes contain samples from different animals, and each lane was loaded with 20 μg protein; 4–7 animals were assayed for each Kv channel α subunit protein.

Article Snippet: Incubations with primary antibodies (all purchased from Alomone Labs, Jerusalem: Kv1.1 (cat. #AN-03), Kv1.2 (cat. #AN-04), Kv1.3 (cat. #AN-03), Kv1.4 (cat. #AN-06), Kv1.5 (cat. #AN-04), Kv1.6 (cat. #AN-03), Kv4.2 (cat. #AN-04) and Kv4.3 (cat. #AN-04)) at dilutions of 1:200-500 were performed in Tris-saline solution containing nonfat milk at 0.05% (w/v), Tween 20 at 0.05% (v/v) and sodium azide at 0.02% (w/v) for 1.5 hours at room temperature with rocking.

Techniques: Western Blot

Journal: Reproductive biology and endocrinology : RB&E

Article Title: The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

doi: 10.1186/1477-7827-5-41

Figure Lengend Snippet: Time course of loss of 4-AP responsiveness and Kv4.3 expression in pregnant mouse myometrium . Myometrial samples were obtained from nonpregnant (NP, n = 5), 7 days pregnant (P7, n = 3), 10 days pregnant (P10, n = 3), 15 days pregnant (P15, n = 4) and term-pregnant (P20, n = 5) mice. Panel A: Results from non-cumulative concentration-response experiments are presented as mean percentages of the maximal response to 4-AP in NP tissues. Panel B: Densitometry of Kv4.3 expression in the same samples evaluated in panel (A) and typical Western blot results demonstrating expression of Kv4.3 as well as Kv1.4 and γ-actin to confirm equal protein loading. Band densities are expressed in graph as a percentage of the maximal band density on each film; asterisks denote a significant difference (P = 0.01) between average NP and term-pregnant sample band densities.

Article Snippet: Incubations with primary antibodies (all purchased from Alomone Labs, Jerusalem: Kv1.1 (cat. #AN-03), Kv1.2 (cat. #AN-04), Kv1.3 (cat. #AN-03), Kv1.4 (cat. #AN-06), Kv1.5 (cat. #AN-04), Kv1.6 (cat. #AN-03), Kv4.2 (cat. #AN-04) and Kv4.3 (cat. #AN-04)) at dilutions of 1:200-500 were performed in Tris-saline solution containing nonfat milk at 0.05% (w/v), Tween 20 at 0.05% (v/v) and sodium azide at 0.02% (w/v) for 1.5 hours at room temperature with rocking.

Techniques: Expressing, Concentration Assay, Western Blot

Journal: PLoS ONE

Article Title: Electrical Remodeling of Preoptic GABAergic Neurons Involves the Kv1.5 Subunit

doi: 10.1371/journal.pone.0096643

Figure Lengend Snippet: A. qPCR analysis of Kv1.1, 1.3, 1.4 and 1.5 expression in preoptic area from GAD65-GFP and Kv4.2−/−;GAD65-GFP mice (n = 6 mice each). For normalization GAPDH was selected as housekeeping gene. Bars represent averaged normalized concentrations ±SD. ** represents significant differences between GAD65-GFP and Kv4.2−/−;GAD65-GFP groups P<0.01 (t-test). B. Western blot analysis of the expression of Kv1.5 subunits. (A) Lysates prepared from the preoptic area of GAD65-GFP or Kv4.2−/−;GAD65-GFP mice (n = 6 animals for each genotype) were fractionated, transferred to PVDF membranes and probed with a specific anti-Kv1.5 antibody. Blots were also probed with antibodies against GAPDH to confirm equal loading of proteins. In each lane, anti-Kv1.5 antibody signals were quantified and normalized to the anti-GAPDH antibody signals. C. In Kv4.2−/−;GAD65-GFP preoptic area, the mean ±S.D. expression levels of Kv1.5 protein were significantly (**, p<0.01) higher than in GAD65-GFP preoptic area. Molecular masses are indicated on the blots by arrows.

Article Snippet: The polyclonal Kv1.5 antibody (dilution 1∶500) was from Alomone labs (#APC-004).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: HUVECs were incubated with AngII at different concentrations for different times. Ang II time (A)- or concentration (B)-dependently enhanced the Kv1.5 protein expression. Pretreatment with MT for 30 min inhibited Ang II (2 µM, 24 h)-induced increase in intracellular ROS levels in HUVECs in a concentration-dependent manner (C), as determined by DCF fluorescence. MT also significantly attenuated Ang II (2 µM, 24 h)-induced HUVEC injury. Incubation of HPAECs with oxLDL at the concentration of 37.5, 75 and 150 µg/ml significantly increased EC injury (D), intracellular ROS production (E) and Kv1.5 protein expression (F) in a concentration-dependent manner. The values were presented as ± SEM of 6 independent experiments for Ang II- or oxLDL-treatment, respectively. * P <0.05, ** P <0.01 vs. control; $ P <0.05, # P <0.01 vs. Ang II group.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Incubation, Concentration Assay, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: ( A ). HPAECs were treated with 2.5, 5, 10 nM Kv1.5 siRNA for 48 h and Kv1.5 protein expression was analyzed by western blot. * P <0.05, ** P <0.01 vs. Vehicle control, n = 3. ( B ). HPAECs were infected with adnovirus containing KCNA5 (Ad-Kv1.5) and control–adnovirus (Ad-con) for 12, 24, 48 h. * P <0.05, ** P <0.01 vs. Ad-con at corresponding time points, n = 3.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Expressing, Western Blot, Infection

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: HPAECs were transfected with Kv1.5 siRNA (10 nM) or control siRNA for 48 h, or infected with adnovirus containing KCNA5 and control–adnovirus for 24 h, then treated with oxLDL (150 µg/ml) for further 24 h. The oxLDL-induced EC injury was detected by optical microscopy and DAPI staining. The optical microscope observation (A) and DAPI staining (B) show the morphological changes in Kv1.5 siRNA pretreated HPAECs. The mean values of percentages of apoptotic cells were summarized after DAPI staining in Kv1.5 siRNA (C)- and KCNA5 adnovirus- pretreated (D) HPAECs. The values are presented as means ± SEM of 6 independent experiments. * P <0.05, ** P <0.01 vs. Vehicle control; # P <0.05, ## P <0.01 vs. Ad-con.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Transfection, Infection, Microscopy, Staining

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: Intracellular ROS levels were detected using DCFH-DA staining in HPAECs transfected with Kv1.5 siRNA (10 nM) or infected with adnovirus containing KCNA5 , which were treated with oxLDL (150 µg/ml) for further 1 h. ( A ) & ( B ). Kv1.5 siRNA significantly attenuated oxLDL-induced endothelial ROS overproduction in HPAECs, as demonstrated by representative images from laser scanning confocal microscopy and bar graph showing fluorescence intensity measured in Multi-Mode Microplate Reader. ( C ). Bar graph shows that adnoviral KCNA5 gene transfer exaggerated oxLDL-induced endothelial ROS overproduction in HPAECs. The values are presented as means ± SEM of 6 independent experiments. * P <0.05, ** P <0.01 vs. Vehicle control; # P <0.05, ## P <0.01 vs. Ad-con.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Staining, Transfection, Infection, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: The mitochondrial ROS generation was detected using MitoSOX reagents in HPAECs transfected with Kv1.5 siRNA (10 nM) or infected with adnovirus containing KCNA5 , which were treated with oxLDL (150 µg/ml) for further 1 h. ( A ) The representative images from laser scanning confocal microscopy Kv1.5 siRNA significantly attenuated oxLDL-induced mitochondrial superoxide production in HPAECs. ( B ) & ( C ). The mitochondrial ROS levels in HPAECs transfected with Kv1.5 siRNA (B) or infected with adnovirus overexpression of Kv1.5 (C) were quantified by the fluorescent plate reader and expressed as the ratio of MitoSOX to Mitotracker. The values are presented as means ± SEM of 6 independent experiments. * P <0.05, ** P <0.01 vs. Vehicle control; # P <0.05, ## P <0.01 vs. Ad-con.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Transfection, Infection, Confocal Microscopy, Over Expression

Journal: PLoS ONE

Article Title: Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

doi: 10.1371/journal.pone.0049758

Figure Lengend Snippet: The UCP2 protein expression was detected by western blot in HPAECs transfected with Kv1.5-siRNA (A) and or infected with adnovirus containing KCNA5 (B). Digital photographs showing UCP2 protein expression are on the top panel and bar graph showing quantitative analysis of the total protein are on the lower panel. The values are presented as means ± SEM of 6 independent experiments. ** P <0.01 vs. corresponding control group; # P <0.01 vs. oxLDL group.

Article Snippet: The membranes were probed with antibodies against Kv1.5 (Alomone Labs, USA) or UCP2 (Biolegend, USA), respectively.

Techniques: Expressing, Western Blot, Transfection, Infection

Journal: PLoS ONE

Article Title: SAP97 and Cortactin Remodeling in Arrhythmogenic Purkinje Cells

doi: 10.1371/journal.pone.0106830

Figure Lengend Snippet: Co-immunolocalization of Kv1.5 (green) and SAP-97(red) in an aggregate of NZPCs.

Article Snippet: The antibodies used in this study include rabbit anti-Kv1.5 (1∶100, Alomone labs), mouse anti-SAP97 (1∶100, Santa Cruz), mouse anti-cortactin (1∶200, Millipore), rabbit anti-cortactin (1∶100, Cell Applications), and mouse anti-N-cadherin (1∶100, BD).

Techniques:

Journal: PLoS ONE

Article Title: SAP97 and Cortactin Remodeling in Arrhythmogenic Purkinje Cells

doi: 10.1371/journal.pone.0106830

Figure Lengend Snippet: B , Costaining of the subcellular surface for Kv1.5 (green) and SAP-97 (red) in an NZPC. C , Localization of Kv1.5 (green) and SAP97 (red) in the cell ID area of an NZPC. D . Analyses of the merged image of Figure 3B illustrating the lack of true colocalization between SAP-97 (red) and Kv1.5 (green).Note the near absence of yellow signals. Scales were inset.

Article Snippet: The antibodies used in this study include rabbit anti-Kv1.5 (1∶100, Alomone labs), mouse anti-SAP97 (1∶100, Santa Cruz), mouse anti-cortactin (1∶200, Millipore), rabbit anti-cortactin (1∶100, Cell Applications), and mouse anti-N-cadherin (1∶100, BD).

Techniques:

Journal: PLoS ONE

Article Title: SAP97 and Cortactin Remodeling in Arrhythmogenic Purkinje Cells

doi: 10.1371/journal.pone.0106830

Figure Lengend Snippet: C , Enlarged image showing the colocalization of Kv1.5 and SAP-97 in IDs. D , Enlarged image showing the localization of Kv1.5 (green) and SAP-97 (red) in the center of an IZPC. E , Enlarged image showing the localization of Kv1.5 (green) and SAP-97 (red) at the subcellular surface of an IZPC. Compare to . Scales were inset.

Article Snippet: The antibodies used in this study include rabbit anti-Kv1.5 (1∶100, Alomone labs), mouse anti-SAP97 (1∶100, Santa Cruz), mouse anti-cortactin (1∶200, Millipore), rabbit anti-cortactin (1∶100, Cell Applications), and mouse anti-N-cadherin (1∶100, BD).

Techniques: