Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 1. Co-immunoprecipitation of TRPV4, TRPC1 and KCa1.1(α) in the lysates of human LIMA VSMCs. A and B, TRPC1 with KCa1.1(α). C and D, TRPC1 with TRPV4. E and F, TRPV4 with KCa1.1(α). The data are representative of 3 to 4 experiments. The pulling antibody and the blotting antibody were indicated (+). Control immunoprecipitation (−) was performed using the preimmune IgG. IP, immunoprecipitation; Blot, immunoblot.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Immunoprecipitation, Control, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 5. Membrane potential changes in HEK293 cells that over-expressed with TRPV4, TRPC1 and KCa1.1. A and B, representative traces illustrating the hyperpolarizing responses to 300 nmol/L 11,12-EET (A), 5 μmol/L 4a-PDD (B) and 100 nmol/L GSK1016790A (C). D–F, summary of data showing the maximal hyperpolarization to 300 nmol/L 11,12-EET (D), 5 μmol/L 4a- PDD (E) and 100 nmol/L GSK1016790A (F) under different conditions. C1mut-pore, TRPC1 were replaced by a TRPC1 pore-dead mutant C1mut-pore. Values are means ± SE (n = 5 to 9). **, P b 0.01 compared to the controls (non-transfected); ##, P b 0.01 compared to the transfected cells without inhibitors; $$, P b 0.01 compared to preimmune IgG.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Membrane, Mutagenesis, Transfection

Journal: Biochimica et biophysica acta

Article Title: Epoxyeicosatrienoic acids act through TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle membrane hyperpolarization and relaxation in human internal mammary arteries.

doi: 10.1016/j.bbadis.2014.12.010

Figure Lengend Snippet: Fig. 4. Requirement of TRPC1 for TRPV4 and KCa1.1 interaction. A–D, co-immunoprecipitation of TRPV4 and KCa1.1(α) in the lysates of HEK293 cells. A and B, HEK293 cells that over- expressed with TRPV4, TRPC1 and KCa1.1. C and D, HEK293 cells that over-expressed with TRPV4 and KCa1.1 but without TRPC1. The right lanes in C and D were immunoblots using cell lysates without immunoprecipitation. The pulling antibody and the blotting antibody were indicated (+). Control immunoprecipitation (−) was performed using the preimmune IgG. IP, immunoprecipitation; Blot, immunoblot. n = 3 to 4 experiments. E–F, the maximal hyperpolarization in response to 300 nmol/L 11,12-EET (E) and 5 μmol/L 4α-PDD (F) in HEK293 cells that were transfected with indicated constructs. C1 stands for TRPC1. V4 stands for TRPV4. Values are means ± SE (n = 5 to 9). **P b 0.01 compared to the controls (non-transfected), ##P b 0.01 compared to C1 + V4 + KCa1.1.

Article Snippet: The primary rabbit antibodies against KCa1.1(α) (APC-021), TRPC1 (ACC-010), and TRPV4 (ACC-034) were from Alomone Labs. T1E3 was raised by us from rabbits.

Techniques: Immunoprecipitation, Western Blot, Control, Transfection, Construct

Journal: Acta pharmacologica Sinica

Article Title: The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane.

doi: 10.1038/aps.2014.165

Figure Lengend Snippet: Figure 2. Effects of atazanavir (ATV) on human ether-a-go-go-related gene (hERG) potassium (K+) currents expressed in HEK293 cells. (A) Original traces of hERG K+ currents elicited by the pulse protocol shown in the inset under control conditions (left) and 5 min after incubation with 10 µmol/L ATV (right). (B and C) I–V relationships for (B) currents measured at the end of depolarizing steps and (C) tail currents in control cells and in cells exposed to 10 µmol/L ATV (bP<0.05, cP<0.01 vs control, n=6, mean±SD). (D) Peak tail currents were normalized to their respective maximum current amplitude (control and drugs) to illustrate changes in half-maximal activation voltages. (E) Representative current traces showing steady-state inactivation using the pulse protocol (upper panel).

Article Snippet: The membranes were blocked with 5% non-fat dry milk before incubation with rabbit anti-hERG antibody (1:400, APC-109; Alomone Labs, Jerusalem, Israel) overnight.

Techniques: Control, Incubation, Activation Assay

Journal: Acta pharmacologica Sinica

Article Title: The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane.

doi: 10.1038/aps.2014.165

Figure Lengend Snippet: Figure 5. Double immunofluorescence staining of human ether-a-go-go-related gene (hERG) protein and the endoplasmic reticulum (ER) marker protein calreticulin in HEK293 cells incubated in normal (A) or 30 μmol/L ATV-containing medium (B) for 36–48 h.

Article Snippet: The membranes were blocked with 5% non-fat dry milk before incubation with rabbit anti-hERG antibody (1:400, APC-109; Alomone Labs, Jerusalem, Israel) overnight.

Techniques: Double Immunofluorescence Staining, Marker, Incubation

Journal: Acta pharmacologica Sinica

Article Title: The protease inhibitor atazanavir blocks hERG K(+) channels expressed in HEK293 cells and obstructs hERG protein transport to cell membrane.

doi: 10.1038/aps.2014.165

Figure Lengend Snippet: Figure 6. Western blot analysis of cells expressing human ether-a-go-go-related gene (hERG) Y652A and F656C mutant channels. Cells expressing hERG Y652A and F656C mutant channels were analyzed by Western blot under control conditions and after 36–48 h incubation with ATV (1.0, 10, and 30 µmol/L). bP<0.05 compared with control. n=4 per group.

Article Snippet: The membranes were blocked with 5% non-fat dry milk before incubation with rabbit anti-hERG antibody (1:400, APC-109; Alomone Labs, Jerusalem, Israel) overnight.

Techniques: Western Blot, Expressing, Mutagenesis, Control, Incubation

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 3. Responsive activation of TRPV1-mediated intracellular Ca2+ concentration. A) CLSM images of 143B and HOS cells in various groups after using the Fluo-4 probe to monitor Ca2+ influx. The scale bar is 20 μm. B,C) FC quantitative value of Ca2+ uptake in 143B and HOS cells after different treatments. D) The protein expression level of TRPV1 was determined by western blot analysis. E) Statistical analyses of TRPV1 (the data are presented as the mean ± SD, n = 3, ***p < 0.001).

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Activation Assay, Concentration Assay, Expressing, Western Blot

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 4. Evaluation of ferroptosis. The production of Lipid-ROS in A) 143B and B) HOS cells after different interventions as measured by CLSM. The scale bars represent 20 μm. C) FC analysis of Lipid-ROS after different interventions in 143B and HOS cells. D,E) Confocal images and FC analyses of ROS generation in 143B and HOS cells treated with different treatments and stained with DCFH-DA (scale bar = 20 μm). F,G) Quantitative value of intracellular C11-BODIPY and DCFH-DA FL intensity (the data are presented as the mean ± SD, n = 3, ***p < 0.001). H) The morphology of mitochondria after various treatments as observed by TEM. The scale bars represent 2 μm. I) The expression level of Gpx4 protein was measured by western blot analysis. J) The expression of TRPV1 and Gpx4 by IF observation after different treatments in 143B cells (scale bar = 100 μm).

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Staining, Expressing, Western Blot

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 5. A) In MG63, HOS, and 143B cells, the effects of CAP on the intracellular oxygen level after different treatments were observed and analyzed by confocal microscopy (scale bar = 20 μm) and FC. B) Quantitative value of intracellular Image-iT Green Hypoxia Reagent FL intensity (the data are presented as the mean ± SD, n = 3, ***p < 0.001). C,D) The expression level of HIF-1𝛼protein was analyzed by western blot (the data are presented as the mean ± SD, n = 3, ***p < 0.001). E,F) The expression of TRPV1 and HIF-1𝛼by IF observation after different treatments in 143B and HOS cells (scale bar = 100 μm).

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Confocal Microscopy, Expressing, Western Blot

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Capsaicin Enhanced the Efficacy of Photodynamic Therapy Against Osteosarcoma via a Pro-Death Strategy by Inducing Ferroptosis and Alleviating Hypoxia.

doi: 10.1002/smll.202306916

Figure Lengend Snippet: Figure 8. Evaluation and biosafety of different treatments. A) H&E staining and images of PCNA, Ki67, TRPV1, Gpx4, and HIF-1𝛼. IHC staining in tumor specimens after various treatments (scale bar = 100 μm). B–F) IOD values in various groups were analyzed by processing IHC staining through ImagePro Plus. G) Triple IF staining of TRPV1 (red), Gpx4 (orange), and HIF-1𝛼(green) in tumor specimens after various treatments (scale bar = 50 μm). H) TUNEL staining of tumor sections after various treatments (scale bar = 50 μm). I) H&E staining of major organs of mice after different treatments (scale bar = 100 μm). J) Hematological and blood biochemical tests of mice after various treatments.

Article Snippet: Mouse anti-human TRPV1 (cat. 66983-1-Ig), mouse anti-human NQO1 (cat. 67240-1-Ig), rabbit anti-human GCLM (cat. 14241-1-AP), rabbit anti-human PCNA (cat. 10205-2-AP), rabbit anti-human KI67 (cat. 27309-1-AP), and rabbit antihuman HIF-1α (cat. 20960-1-AP) were acquired from Proteintech Corp (USA).

Techniques: Staining, Immunohistochemistry, TUNEL Assay

Journal: Scientific Reports

Article Title: Syntabulin regulates the trafficking of PICK1-containing vesicles in neurons

doi: 10.1038/srep20924

Figure Lengend Snippet: ( A ) Neurons were infected with syntabulin shRNA lentivirus as indicated at DIV5 and examined at DIV13. Syntabulin knockdown significantly increased total ASIC2 expression but reduced surface ASIC2 levels as compared with the levels in control and scrambled-shRNA control. ( B ) Quantification of normalized ASIC2/GAPDH ratio, total ASIC2/surface ASIC2 ratio and surface ASIC2 levels. One-way ANOVA and Sidak test; ** P = 0.008, and P = 0.009 for Stb sh#1 Total ASIC2/GAPDH and Surface ASIC2/GAPDH compared to control respectively; * P = 0.044 and * P = 0.016 for Stb #1 total ASIC2/GAPDH and Surface/Total ASIC2 ratio compared to scrambled shRNA control respectively; * P = 0.020 for Stb #1 Surface/Total ASIC2 ratio compared to control; “ns” P > 0.05. n = 4 − 6 independent experiments. Error bars represent the SEM. ( C , D ) Hoechst staining of neurons after syntabulin knockdown, in pH 7.4 solution and pH6.0 solution. Knockdown of syntabulin significantly increased cell death as compared with controls in pH6.0. Scale bar = 20 μm. ( E ) Quantification of normalized total Hoechst intensity. One-way ANOVA and Sidak test; * P = 0.035 compared to control; * P = 0.043 compared to scrambled-shRNA control; “ns” P > 0.05; n = 6 independent experimental repeats. Error bars represent the SEM.

Article Snippet: The following antibodies were purchased: rabbit polyclonal ASIC2 antibody, Proteintech (17851-1-AP); mouse myc and β-tubulin antibodies, DSHB (9E10 and E7); mouse GAPDH antibody, Beyotime (GA019); mouse Map2 antibody, Sigma (M9942); and mouse β-actin antibody, Sigma (A5316).

Techniques: Infection, shRNA, Knockdown, Expressing, Control, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Specific primers of KCNK channel genes for qPCR.

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Expression profiles of KCNK family members in PASMCs from IPAH patients. The expression of KCNK channel family members (KCNK1 to 18, except for 8, 11, and 14) in normal- and IPAH-PASMCs was examined by qPCR and Western blotting. ( A ) Expression of KCNK family members in normal- and IPAH-PASMCs at the mRNA level ( n = 4). The mRNA expression level of KCNK was normalized to that of β-actin. Inset , the expression ratios of KCNK1, 2, 3, and 6 in IPAH-PASMCs to normal-PASMCs ( n = 4). ( B ) Protein expression of KCNK1, 2, 3, and 6 channels in normal- and IPAH-PASMCs ( n = 6–8). The protein expression of KCNK channels was normalized to that of β-actin and normal-PASMCs. Note that the expression of KCNK1/TWIK1 and KCNK2/TREK1 was up-regulated, whereas that of KCNK3/TASK1 and KCNK6/TWIK2 was down-regulated in IPAH-PASMCs. Data are presented as means ± S.E. * p < 0.05, ** p < 0.01 vs. normal-PASMCs (Mann–Whitney U test).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Up-regulated expression of KCNK1 and KCNK2 channels in PASMs from MCT-PH rats. The protein expression of KCNK1 and KCNK2 channels in PASMs from MCT-PH rats was examined by Western blotting and immunohistochemical staining. ( A,B ) Protein expression of KCNK1 ( A ) and KCNK2 ( B ) channels in PASMCs from control and MCT-PH rats ( n = 8). Protein expression was normalized to that of β-actin and the control group. ( C,D ) Representative immunohistochemical images of the lung sections of control and MCT-PH rats stained with a KCNK1 ( C ; green ), KCNK2 ( D ; green ), or α -SMA ( red ) antibody. Cell nuclei were stained with DAPI ( blue ). Similar results were obtained from six independent experiments. Note that the expression of KCNK1/TWIK1 and KCNK2/TREK1 was up-regulated in PASMs from MCT-PH rats. Data are presented as means ± S.E. ** p < 0.01, *** p < 0.001 vs. the control (Mann–Whitney U test).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining, Control, MANN-WHITNEY

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Expression of KCNK1 and KCNK2 channels in PASMs from SuHx-PH rats and hypoxia-PH mice. The protein expression of KCNK1 and KCNK2 channels in PASMs from SuHx-PH rats and hypoxia-PH mice was examined by Western blotting. ( A,B ) Protein expression of KCNK1 ( A ) and KCNK2 ( B ) channels in PASMs from control and SuHx-PH rats ( n = 6). Protein expression was normalized to that of β-actin and the control group. ( C,D ) Protein expression of KCNK1 ( C ) and KCNK2 ( D ) channels in PASMs from normoxia and hypoxia-PH mice ( n = 6). Protein expression was normalized to that of β-actin and the normoxia group. Data are presented as means ± S.E. * p < 0.05, ** p < 0.01 vs. the control or normoxia group (Mann–Whitney U test).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Expressing, Western Blot, Control, MANN-WHITNEY

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: siRNA knockdown of KCNK1 and KCNK2 channels in IPAH-PASMCs. The effects of KCNK1 and KCNK2 siRNAs on the expression of KCNK1, KCNK2, KCNK3, and KCNK6 channels in IPAH-PASMCs were examined by qPCR and Western blotting. ( A,B ) Knockdown efficiency at the mRNA level of siRNA targeting KCNK1 ( A ) or KCNK2 ( B ) in IPAH-PASMCs ( n = 6). mRNA expression was normalized to that of β-actin and control siRNA. ( C,D ) Knockdown efficiency at the protein level of siRNA targeting KCNK1 ( C ) or KCNK2 ( D ) in IPAH-PASMCs ( n = 6). Protein expression was normalized to that of β-actin and control siRNA. Data are presented as means ± S.E. ** p < 0.01 vs. control siRNA (Mann–Whitney U test).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Knockdown, Expressing, Western Blot, Control, MANN-WHITNEY

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Contribution of KCNK1 and KCNK2 channels to the proliferation and migration of IPAH-PASMCs. The involvement of KCNK1 and KCNK2 channels in the proliferation and migration of IPAH-PASMCs was examined by siRNA knockdown methods. ( A,B ) Inhibitory effects of the transfection with KCNK1 ( A ) or KCNK2 ( B ) siRNA for 48 h on the growth of IPAH-PASMCs using the Cell Counting Kit-8 assay ( n = 4). Absorbance was normalized by control siRNA. ( C,D ) Inhibitory effects of the transfection with KCNK1 ( C ) or KCNK2 ( D ) siRNA for 48 h on the excessive proliferation of IPAH-PASMCs using the BrdU incorporation assay ( n = 4). Absorbance was normalized by control siRNA. ( E ) Anti-migratory effects of the transfection with KCNK1 or KCNK2 siRNA on the migration of IPAH-PASMCs for 24 h using the Transwell assay ( n = 4). Data are presented as means ± S.E. * p < 0.05, ** p < 0.001 vs. control siRNA (Student's t -test ( A–D ) or Steel's test following Kruskal-Wallis test ( E )).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Migration, Knockdown, Transfection, Cell Counting, Control, BrdU Incorporation Assay, Transwell Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Contribution of KCNK1 and KCNK2 channels to the resting membrane potential and [Ca 2+ ] cyt in IPAH-PASMCs. The effects of the siRNA knockdown of KCNK1 and KCNK2 on the resting membrane potential and [Ca 2+ ] cyt were measured in IPAH-PASMCs. Membrane potential was monitored with the voltage-sensitive fluorescent indicator, DiBAC 4 (3). Fluorescent intensity of DiBAC 4 (3) (F/F 140K ) was increased and decreased by membrane depolarization and hyperpolarization, respectively. Fluorescent intensity signal was normalized by the maximum fluorescent intensity in the 140-mM K + HEPES-buffered solution (theoretically 0 mV). [Ca 2+ ] cyt (F 340 /F 380 ) was measured using the Ca 2+ -sensitive fluorescent indicator, fura-2/AM. ( A ) Time courses of the membrane potential in IPAH-PASMCs transfected with control ( n = 115), KCNK1 ( n = 131), or KCNK2 ( n = 98) siRNA before and after the perfusion with 140 mM K + HEPES-buffered solution. ( B ) Summarized data of the resting membrane potential in control ( n = 115), KCNK1 ( n = 131), or KCNK2 ( n = 98) siRNA-treated IPAH-PASMCs. The resting membrane potential was defined as the average value of F/F 140K for 5 min before the perfusion with 140 mM K + HEPES-buffered solution. ( C ) Resting [Ca 2+ ] cyt levels in IPAH-PASMCs transfected with control ( n = 73), KCNK1 ( n = 63), or KCNK2 ( n = 48) siRNA. ( D ) Summarized data of the resting [Ca 2+ ] cyt in control ( n = 73), KCNK1 ( n = 63), or KCNK2 ( n = 48) siRNA-treated IPAH-PASMCs. The resting [Ca 2+ ] cyt was defined as the average value of F 340 /F 380 for 5 min after the beginning of the experiment. Data are presented as means ± S.E. *** p < 0.001 vs. control siRNA (Scheffé's test following ANOVA).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Membrane, Knockdown, Transfection, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Up-regulated expression of two-pore domain K + channels, KCNK1 and KCNK2, is involved in the proliferation and migration of pulmonary arterial smooth muscle cells in pulmonary arterial hypertension

doi: 10.3389/fcvm.2024.1343804

Figure Lengend Snippet: Effects of KCNK1 and KCNK2 channel knockdown on the phosphorylation of JNK in IPAH-PASMCs. The expression and phosphorylation levels of JNK in normal- and IPAH-PASMCs and the effects of the siRNA knockdown of KCNK1 and KCNK2 channels were examined by Western blotting. ( A ) The phosphorylation levels of JNK in normal- and IPAH-PASMCs ( n = 6). Protein expression was normalized to that of β-actin and normal-PASMCs. ( B ) The effects of the siRNA knockdown of KCNK1 channels on the phosphorylation levels of JNK in IPAH-PASMCs ( n = 6). Protein expression was normalized to that of β-actin and control siRNA. ( C ) The effects of the siRNA knockdown of KCNK2 channels on the phosphorylation levels of JNK in IPAH-PASMCs ( n = 6). Protein expression was normalized to that of β-actin and control siRNA. Data are presented as means ± S.E. ** p < 0.01, ** p < 0.01 vs. normal-PASMCs or control siRNA (Mann–Whitney U test).

Article Snippet: The membrane was blocked with Tris-buffered saline containing bovine serum albumin (5%) and Tween 20 (0.1%; MilliporeSigma) at room temperature (25°C) for 3 h and then treated with a primary antibody for KCNK1 (1:800; APC-110), KCNK2 (1:800; APC-047), KCNK3 (1:800; APC-024), KCNK6 (1:800; APC-040, Alomone Labs, Jerusalem, Israel), JNK (1:1000; #9252), or phospho ( p )-JNK (1:1000; #4668, Cell Signaling Technology, Danvers, MA, USA) at 4°C for 18 h. Immunoblotted membranes were then exposed to an anti-rabbit HRP-conjugated IgG secondary antibody (1:5000; #170-6515, Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 1 h. Blotting signals were detected using an ImmunoStar LD reagent (Fujifilm Wako Pure Chemical) and observed with the Imager 600 system (GE HealthCare Technologies, Chicago, IL, USA).

Techniques: Knockdown, Phospho-proteomics, Expressing, Western Blot, Control, MANN-WHITNEY

Journal: Pharmaceuticals

Article Title: Segmental Upregulation of ASIC1 Channels in the Formalin Acute Pain Mouse Model

doi: 10.3390/ph15121539

Figure Lengend Snippet: ASIC1 levels in the pain pathway. ( A ) Representative membrane of lysates of ACC tissue from formalin (For)- or vehicle (Veh)- injected male mice at Ip (ipsilateral) or Con (contralateral) sides to the injection detected using ASIC1 and tubulin antibodies (left), and plot of the results obtained from membranes for ASIC1/tubulin detected levels (right) (4–5 animals used per condition). ( B ) Representative membrane of lysates of SC tissue from For- or Veh- injected mice at different lumbar (3, 4, 5) levels detected using ASIC1 and tubulin antibodies (left), and plot of the results obtained from membranes for ASIC1/tubulin detected levels (right) (4–5 animals used per condition). Notice the gradient decrease from level 3 to 5. ( C ) Representative membrane of lysates of a pool of DRG tissue from different lumbar (3, 4, 5) levels in For- or Veh- injected male mice at Ip or Con sides to the injection detected using ASIC1 and tubulin antibodies. Notice the same gradient pattern as in ( B , D ) Images of the regions dissected (SC, DRG segments) used for the experiments in ( B , C ). One-way ANOVA, **** p < 0.0001; *** p < 0.001 ** p < 0.01. Scale bar 5 mm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti ASIC1 (Alomone ASC-014, 1:1000); mouse monoclonal anti-tubulin (DM1a; Cell signaling #3873, 1:5000); rabbit polyclonal anti-total ERK (Santa Cruz, C9, 1:500); rabbit polyclonal anti phosphoERK (Cell Signaling, SC-7383, 1:500).

Techniques: Membrane, Injection

Journal: Pharmaceuticals

Article Title: Segmental Upregulation of ASIC1 Channels in the Formalin Acute Pain Mouse Model

doi: 10.3390/ph15121539

Figure Lengend Snippet: Schematic representation of ASIC1 upregulation in ACC, SC and DRG in the formalin mouse model of pain. At the cortex, the ACC contralateral to the injection (left hind paw) shows a higher ASIC1 protein level. At the SC and DRGs, there is a gradient decreasing from L3 to L5 lumbar segments. Spinal nerves contribution to the sciatic nerve is represented with lines, thicker for those contributing to a greater extent. ASIC1 protein levels are represented showing an increase with more intense color. (IP, ipsilateral; CON, contralateral; ACC, Anterior Cingulate Cortex; DRG (Dorsal Root Ganglia; SC, Spinal Cord; L3, 4, 5, lumbar 3, 4, 5).

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti ASIC1 (Alomone ASC-014, 1:1000); mouse monoclonal anti-tubulin (DM1a; Cell signaling #3873, 1:5000); rabbit polyclonal anti-total ERK (Santa Cruz, C9, 1:500); rabbit polyclonal anti phosphoERK (Cell Signaling, SC-7383, 1:500).

Techniques: Injection