Review

dctn2 cdna (OriGene)

OriGene is a verified supplier

OriGene manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

OriGene dctn2 cdna
Dctn2 Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/dctn2+cdna/Dctn2+(NM_027151)+Mouse+Untagged+Clone/pm24912985-61-2-25
Average 90 stars, based on 2 article reviews

dctn2 cdna - by Bioz Stars, 2026-09

90/100 stars

Images

other:

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.
Article Snippet: Nucleofection of DCTN2 cDNA and Nox1 cDNA Both dynamitin (Dynactin 2: DCTN2) cDNA (Catalog no. MC200162) and Nox1cDNA plasmids (Catalog no. MG226022) were purchased from OriGene Technologies.

Image Search Results

Dynein binds to the Kv7.4 C terminus. (A) Amino acid alignment of the end of the c-helix of Xenopus Kv7.1 (used for the modeling in B) and human Kv7.1–Kv7.5 channels. (B) Close view of the Kv7 channel c-helix using the structure of Xenopus Kv7.1 suggests that several residues (highlighted in green) in the dynein-binding motifs indeed are accessible to the intracellular environment. Those in red are not exposed to the intracellular environment and are therefore unlikely to be required for dynein recognition. (C, i–iii) Representative whole-cell voltage clamp recordings and respective I-V relations compared between Kv7.4 and Kv7.4-Q580A (i); when cotransfected with p50/dynamitin (ii); or incubation with 3 µM ciliobrevin D (iii). Statistical comparisons were made with a two-way ANOVA, followed by a Bonferroni multiple comparisons test, where P < 0.05, P < 0.01, and P < 0.001 are depicted by *, **, and ***, respectively. (C iv) Mean V 1/2 for steady-state activation was compared for each condition with a one-way ANVOA. (D) Docking simulations performed with SwissDock on Kv7.1, Kv7.4, Kv7.1-Q560A, and Kv7.4-Q580A showing ciliobrevin D binding to both mutant channels but neither WT channel. Each of the four spirals (pink, green, orange, and gold) represent an intercellular C terminus of each of the four Kv7 protein α subunits that multimerize to form a functional channel. Mean values are shown with error bars depicting the SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Dynein binds to the Kv7.4 C terminus. (A) Amino acid alignment of the end of the c-helix of Xenopus Kv7.1 (used for the modeling in B) and human Kv7.1–Kv7.5 channels. (B) Close view of the Kv7 channel c-helix using the structure of Xenopus Kv7.1 suggests that several residues (highlighted in green) in the dynein-binding motifs indeed are accessible to the intracellular environment. Those in red are not exposed to the intracellular environment and are therefore unlikely to be required for dynein recognition. (C, i–iii) Representative whole-cell voltage clamp recordings and respective I-V relations compared between Kv7.4 and Kv7.4-Q580A (i); when cotransfected with p50/dynamitin (ii); or incubation with 3 µM ciliobrevin D (iii). Statistical comparisons were made with a two-way ANOVA, followed by a Bonferroni multiple comparisons test, where P < 0.05, P < 0.01, and P < 0.001 are depicted by *, **, and ***, respectively. (C iv) Mean V 1/2 for steady-state activation was compared for each condition with a one-way ANVOA. (D) Docking simulations performed with SwissDock on Kv7.1, Kv7.4, Kv7.1-Q560A, and Kv7.4-Q580A showing ciliobrevin D binding to both mutant channels but neither WT channel. Each of the four spirals (pink, green, orange, and gold) represent an intercellular C terminus of each of the four Kv7 protein α subunits that multimerize to form a functional channel. Mean values are shown with error bars depicting the SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Binding Assay, Incubation, Activation Assay, Mutagenesis, Functional Assay

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Binding Assay, Incubation, Activation Assay, Mutagenesis, Functional Assay

The Kv7.4-Q580A mutant is less able to bind dynein. (A i) Representative Western blot with HEK293B cells transfected with WT-Kv7.4-EGFP or Q580A-Kv7.4-EGFP showing Kv7.4-EGFP bands (∼104 kD). (A ii) Mean data comparing the Kv7.4-EGFP band intensity in the lysates from HEK293B cells transfected with WT-Kv7.4-EGFP or Q580A-Kv7.4–EGFP. (B i) Representative images of PLAs in HEK293B cells transfected with Kv7.4 (left) or Kv7.4-Q580A (right) using Kv7.4 and dynein antibodies. The EGFP is visualized (green) in the insets to show Kv7.4 expression. Each red punctum is representative of the Kv7 channel and dynein protein localizing within 40 nm of one another. (B ii) Quantification of puncta in HEK293B cells (Kv7.4-WT: 15 cells, Kv7.4-Q580A: 20 cells) with Kv7.4 and dynein and the respective antibodies alone as control, according to an unpaired t test. ***, P < 0.0001. Scale bars, 5 µm. Mean values are shown with error bars depicting the SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: The Kv7.4-Q580A mutant is less able to bind dynein. (A i) Representative Western blot with HEK293B cells transfected with WT-Kv7.4-EGFP or Q580A-Kv7.4-EGFP showing Kv7.4-EGFP bands (∼104 kD). (A ii) Mean data comparing the Kv7.4-EGFP band intensity in the lysates from HEK293B cells transfected with WT-Kv7.4-EGFP or Q580A-Kv7.4–EGFP. (B i) Representative images of PLAs in HEK293B cells transfected with Kv7.4 (left) or Kv7.4-Q580A (right) using Kv7.4 and dynein antibodies. The EGFP is visualized (green) in the insets to show Kv7.4 expression. Each red punctum is representative of the Kv7 channel and dynein protein localizing within 40 nm of one another. (B ii) Quantification of puncta in HEK293B cells (Kv7.4-WT: 15 cells, Kv7.4-Q580A: 20 cells) with Kv7.4 and dynein and the respective antibodies alone as control, according to an unpaired t test. ***, P < 0.0001. Scale bars, 5 µm. Mean values are shown with error bars depicting the SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Mutagenesis, Western Blot, Transfection, Expressing

Dynein colocalizes with Kv7.4 proteins in vascular smooth muscle cells. (A) Representative Western blot showing dynein (∼74 kD) expression in protein lysates from three different samples of rat small mesenteric artery (rMA) and in one protein lysate from untransfected HEK293B cells. (B) Representative structured illumination microscopy images of an isolated smooth muscle cell from a rat small mesenteric artery, stained with antibodies against β-tubulin (green) and dynein (magenta). Scale bar, 10 µm. (C) Representative Kv7.4 bands from a Western blot following coimmunoprecipitation of dynein in HEK293B cells overexpressing Kv7.4. No bands were detected for the nonspecific binding sample (NS) or for the negative control sample (Neg ctr), where normal mouse control IgG was used instead of the specific pulldown antibody. (D) Representative Kv7.4 bands from a Western blot with samples of rat mesenteric artery protein lysate (rMA) that were immunoprecipitated (IP) with a dynein antibody. Each rMA sample contains n = 3 rats’ worth of mesenteric arteries. (E) Representative structured illumination microscopy image of an isolated smooth muscle cell from a rat small mesenteric artery stained with antibodies against Kv7.4 (magenta) and dynein (green). Scale bar, 10 µm. (F i) Representative image of PLAs with Kv7.4 and dynein antibodies in mesenteric artery myocytes. Each red punctum is representative of the Kv7 channel and dynein protein localizing within 40 nm of one another. (F ii) Quantification of puncta in 34 cells ( n = 4 rats) with Kv7.4 and dynein and the respective antibodies alone as control. Mean values are shown with error bars depicting the SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Dynein colocalizes with Kv7.4 proteins in vascular smooth muscle cells. (A) Representative Western blot showing dynein (∼74 kD) expression in protein lysates from three different samples of rat small mesenteric artery (rMA) and in one protein lysate from untransfected HEK293B cells. (B) Representative structured illumination microscopy images of an isolated smooth muscle cell from a rat small mesenteric artery, stained with antibodies against β-tubulin (green) and dynein (magenta). Scale bar, 10 µm. (C) Representative Kv7.4 bands from a Western blot following coimmunoprecipitation of dynein in HEK293B cells overexpressing Kv7.4. No bands were detected for the nonspecific binding sample (NS) or for the negative control sample (Neg ctr), where normal mouse control IgG was used instead of the specific pulldown antibody. (D) Representative Kv7.4 bands from a Western blot with samples of rat mesenteric artery protein lysate (rMA) that were immunoprecipitated (IP) with a dynein antibody. Each rMA sample contains n = 3 rats’ worth of mesenteric arteries. (E) Representative structured illumination microscopy image of an isolated smooth muscle cell from a rat small mesenteric artery stained with antibodies against Kv7.4 (magenta) and dynein (green). Scale bar, 10 µm. (F i) Representative image of PLAs with Kv7.4 and dynein antibodies in mesenteric artery myocytes. Each red punctum is representative of the Kv7 channel and dynein protein localizing within 40 nm of one another. (F ii) Quantification of puncta in 34 cells ( n = 4 rats) with Kv7.4 and dynein and the respective antibodies alone as control. Mean values are shown with error bars depicting the SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Western Blot, Expressing, Microscopy, Isolation, Staining, Binding Assay, Negative Control, Immunoprecipitation

Kv7 channel function is enhanced following dynein inhibition. (A) Representative isometric tension recordings of rat mesenteric artery segments preconstricted with methoxamine (•) before sequentially increasing concentrations of Kv7.2–Kv7.5–specific activator NS15370 were applied in control (left) and ciliobrevin D–treated (right) arteries. (B) Mean concentration-effect curves and EC 50 values to the Kv7.2–Kv7.5–specific activator NS15370 showing the effect of NS15370 in rat mesenteric artery segments before and after 10 µM ciliobrevin D incubation. Mean EC 50 values were compared according to an unpaired t test. **, P < 0.01. (C) 10 µM ciliobrevin D enhanced the relaxation to another Kv7.2–Kv7.5 channel activator, S-1, according to an unpaired t test. *, P < 0.05. (D) Rat mesenteric arteries were transfected with either a Kv7.4-targeted or the control (miss-match) morpholino. Relaxations to S-1 were inhibited in arteries transfected with the Kv7.4-targeted morpholino compared with arteries transfected with the control morpholino. Ciliobrevin D enhanced relaxations to S-1 in control arteries, but ciliobrevin D was unable to enhance S-1–mediated relaxation in Kv7.4 knockdown arteries. A one-way ANOVA followed by a Sidak multiple comparisons test was performed, with ** and *** denoting P < 0.01 and P < 0.001, respectively. Error bars show the mean and SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Kv7 channel function is enhanced following dynein inhibition. (A) Representative isometric tension recordings of rat mesenteric artery segments preconstricted with methoxamine (•) before sequentially increasing concentrations of Kv7.2–Kv7.5–specific activator NS15370 were applied in control (left) and ciliobrevin D–treated (right) arteries. (B) Mean concentration-effect curves and EC 50 values to the Kv7.2–Kv7.5–specific activator NS15370 showing the effect of NS15370 in rat mesenteric artery segments before and after 10 µM ciliobrevin D incubation. Mean EC 50 values were compared according to an unpaired t test. **, P < 0.01. (C) 10 µM ciliobrevin D enhanced the relaxation to another Kv7.2–Kv7.5 channel activator, S-1, according to an unpaired t test. *, P < 0.05. (D) Rat mesenteric arteries were transfected with either a Kv7.4-targeted or the control (miss-match) morpholino. Relaxations to S-1 were inhibited in arteries transfected with the Kv7.4-targeted morpholino compared with arteries transfected with the control morpholino. Ciliobrevin D enhanced relaxations to S-1 in control arteries, but ciliobrevin D was unable to enhance S-1–mediated relaxation in Kv7.4 knockdown arteries. A one-way ANOVA followed by a Sidak multiple comparisons test was performed, with ** and *** denoting P < 0.01 and P < 0.001, respectively. Error bars show the mean and SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Inhibition, Concentration Assay, Incubation, Transfection

Dynein inhibition increases membrane expression of Kv7.4 protein. (A, i–iii) Representative midcell z-section of a mesenteric artery myocyte treated with or without ciliobrevin D and stained for Kv7.4 (top) or NCX (bottom; i). Scale bars, 10 μm. Mean membrane intensity of Kv7.4 ( n = 9; ii) and NCX ( n = 10 or 11; iii) relative to total intensity in cells treated with ciliobrevin D compared with nontreated cells, calculated from midcell z section. Significance was determined by an unpaired t test. **, P < 0.001. (B i) Representative midcell z section of HEK293B cells transfected with Kv7.4-EGFP (green) in nontreated and ciliobrevin D– or colchicine-treated cells. The nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bars, 10 μm. (B ii) Mean membrane intensity of Kv7.4 relative to total intensity in cells treated with ciliobrevin D ( n = 5) or colchicine ( n = 7) compared with nontreated cells ( n = 6), calculated from midcell z section. Significance was determined by a one-way ANOVA. **, P < 0.001; ***, P < 0.0001. Error bars show mean and SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Dynein inhibition increases membrane expression of Kv7.4 protein. (A, i–iii) Representative midcell z-section of a mesenteric artery myocyte treated with or without ciliobrevin D and stained for Kv7.4 (top) or NCX (bottom; i). Scale bars, 10 μm. Mean membrane intensity of Kv7.4 ( n = 9; ii) and NCX ( n = 10 or 11; iii) relative to total intensity in cells treated with ciliobrevin D compared with nontreated cells, calculated from midcell z section. Significance was determined by an unpaired t test. **, P < 0.001. (B i) Representative midcell z section of HEK293B cells transfected with Kv7.4-EGFP (green) in nontreated and ciliobrevin D– or colchicine-treated cells. The nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bars, 10 μm. (B ii) Mean membrane intensity of Kv7.4 relative to total intensity in cells treated with ciliobrevin D ( n = 5) or colchicine ( n = 7) compared with nontreated cells ( n = 6), calculated from midcell z section. Significance was determined by a one-way ANOVA. **, P < 0.001; ***, P < 0.0001. Error bars show mean and SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Inhibition, Expressing, Staining, Transfection

Kv7.4 channels colocalize with caveolin-1. (A) Representative midcell z section of an isolated smooth muscle cell from a rat small mesenteric artery stained with antibodies against caveolin-1 (Cav-1; magenta) and Kv7.4 (green). Scale bar, 10 µm. Insets show the brightfield images of the cells. (B i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with Kv7.4 and caveolin-1 antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). (B ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control: 27 cells, n = 3 rats; M-βCD: 29 cells, n = 3 rats) showing significant decrease of Kv7.4 and caveolin-1 colocalization in M-βCD–treated cells (**, P = 0.008 according to an unpaired t test). (C i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with NCX and caveolin-1 antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). (C ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control, 10 cells; M-βCD, 10 cells) showing equal colocalization of NCX and caveolin-1 in M-βCD–treated cells compared with nontreated cells. Error bars represent SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Kv7.4 channels colocalize with caveolin-1. (A) Representative midcell z section of an isolated smooth muscle cell from a rat small mesenteric artery stained with antibodies against caveolin-1 (Cav-1; magenta) and Kv7.4 (green). Scale bar, 10 µm. Insets show the brightfield images of the cells. (B i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with Kv7.4 and caveolin-1 antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). (B ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control: 27 cells, n = 3 rats; M-βCD: 29 cells, n = 3 rats) showing significant decrease of Kv7.4 and caveolin-1 colocalization in M-βCD–treated cells (**, P = 0.008 according to an unpaired t test). (C i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with NCX and caveolin-1 antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). (C ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control, 10 cells; M-βCD, 10 cells) showing equal colocalization of NCX and caveolin-1 in M-βCD–treated cells compared with nontreated cells. Error bars represent SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Isolation, Staining

Dynein trafficking of Kv7.4 is dependent on cholesterol. (A i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with Kv7.4 and dynein antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). Scale bars, 10 μm. (A ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control, 20 cells, n = 2 rats; M-βCD, 18 cells, n = 2 rats) showing significant decrease of Kv7.4 and caveolin-1 colocalization in M-βCD–treated cells (***, P < 0.0001 according to an unpaired t test). (B i) Representative midcell z section of a mesenteric artery myocyte treated with or without M-βCD and stained for caveolin-1 (Cav-1; magenta) and Kv7.4 or NCX (green). (B ii) Mean membrane intensity of Kv7.4 relative to total intensity in cells increased with M-βCD ( n = 10) compared with nontreated control cells ( n = 8; according to a one-way ANOVA. ***, P < 0.0001). Scale bars, 5 μm. (B iii) Mean membrane intensity of NCX relative to total intensity in cells was equal in M-βCD–treated cells ( n = 10) and nontreated control cells ( n = 9–11). The intensity of caveolin-1 was the same for both groups. Error bars show mean values and SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Dynein trafficking of Kv7.4 is dependent on cholesterol. (A i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with Kv7.4 and dynein antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). Scale bars, 10 μm. (A ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control, 20 cells, n = 2 rats; M-βCD, 18 cells, n = 2 rats) showing significant decrease of Kv7.4 and caveolin-1 colocalization in M-βCD–treated cells (***, P < 0.0001 according to an unpaired t test). (B i) Representative midcell z section of a mesenteric artery myocyte treated with or without M-βCD and stained for caveolin-1 (Cav-1; magenta) and Kv7.4 or NCX (green). (B ii) Mean membrane intensity of Kv7.4 relative to total intensity in cells increased with M-βCD ( n = 10) compared with nontreated control cells ( n = 8; according to a one-way ANOVA. ***, P < 0.0001). Scale bars, 5 μm. (B iii) Mean membrane intensity of NCX relative to total intensity in cells was equal in M-βCD–treated cells ( n = 10) and nontreated control cells ( n = 9–11). The intensity of caveolin-1 was the same for both groups. Error bars show mean values and SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Staining

Cholesterol depletion inhibits Kv7 channel function in mesenteric arteries. (A i) Representative recording of a Kv7.4 current from a Xenopus oocyte before (black) and after (red) application of 1 mM M-βCD. (A ii) Mean data showing the effect of M-βCD on the Kv7.4 current in Xenopus oocytes at 40 mV ( n = 5). **, P < 0.01 according to a paired t test. (B) Representative isometric tension recordings of rat mesenteric artery segments preconstricted with methoxamine (•) before sequentially increasing concentrations of Kv7.2–Kv7.5–specific activator NS15370 were applied in (from left to right) control, M-βCD–treated, cholesterol-saturated M-βCD–treated, and M-βCD + ciliobrevin D–treated arteries. (C) Mean concentration-effect curves and EC 50 values for the Kv7.2–Kv7.5–specific activator NS15370 in isometric tension recordings from either segments of mesenteric artery treated with 5 mM M-βCD (blue, n = 8), 5 mM M-βCD supplemented with cholesterol (green, n = 5), 10 µM ciliobrevin D (red, n = 10), or 5 mM M-βCD + 10 µM ciliobrevin D (orange, n = 6) or from control vessels (black, n = 14) when methoxamine was used to preconstrict the artery segments. M-βCD significantly attenuates the relaxation for NS15370, while cholesterol-saturated M-βCD had no effect compared with control vessels. Ciliobrevin D was unable to enhance relaxation in M-βCD–treated vessels. (D) Mean concentration-effect curves and EC 50 values for the Kv7.2–Kv7.5–specific activator NS15370 in isometric tension recordings from segments of mesenteric artery treated with 3 µM Filipin III. 3 µM Filipin III significantly attenuates relaxation for NS15370. Statistical comparisons on the mean EC 50 values were performed with a one-way ANOVA followed by a Tukey multiple comparisons test. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. Error bars show mean values and SEM.

Journal: The Journal of General Physiology

Article Title: Dynein regulates Kv7.4 channel trafficking from the cell membrane

doi: 10.1085/jgp.202012760

Figure Lengend Snippet: Cholesterol depletion inhibits Kv7 channel function in mesenteric arteries. (A i) Representative recording of a Kv7.4 current from a Xenopus oocyte before (black) and after (red) application of 1 mM M-βCD. (A ii) Mean data showing the effect of M-βCD on the Kv7.4 current in Xenopus oocytes at 40 mV ( n = 5). **, P < 0.01 according to a paired t test. (B) Representative isometric tension recordings of rat mesenteric artery segments preconstricted with methoxamine (•) before sequentially increasing concentrations of Kv7.2–Kv7.5–specific activator NS15370 were applied in (from left to right) control, M-βCD–treated, cholesterol-saturated M-βCD–treated, and M-βCD + ciliobrevin D–treated arteries. (C) Mean concentration-effect curves and EC 50 values for the Kv7.2–Kv7.5–specific activator NS15370 in isometric tension recordings from either segments of mesenteric artery treated with 5 mM M-βCD (blue, n = 8), 5 mM M-βCD supplemented with cholesterol (green, n = 5), 10 µM ciliobrevin D (red, n = 10), or 5 mM M-βCD + 10 µM ciliobrevin D (orange, n = 6) or from control vessels (black, n = 14) when methoxamine was used to preconstrict the artery segments. M-βCD significantly attenuates the relaxation for NS15370, while cholesterol-saturated M-βCD had no effect compared with control vessels. Ciliobrevin D was unable to enhance relaxation in M-βCD–treated vessels. (D) Mean concentration-effect curves and EC 50 values for the Kv7.2–Kv7.5–specific activator NS15370 in isometric tension recordings from segments of mesenteric artery treated with 3 µM Filipin III. 3 µM Filipin III significantly attenuates relaxation for NS15370. Statistical comparisons on the mean EC 50 values were performed with a one-way ANOVA followed by a Tukey multiple comparisons test. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. Error bars show mean values and SEM.

Article Snippet: Kv7.4 and Kv7.4-Q580A were coexpressed with 1 µg p50/dynamitin (catalog: HG14449-UT; SinoBiological).

Techniques: Concentration Assay

Fig. 1 Regulation of dynein ATPase activity by ROS. (A) CAMs were incubated with X/XO (10 lM/0.1 U/ml) or H2O2 (10 lM) for 24 hrs in the presence or absence of dynein inhibitor EHNA (30 lM). Summa- rized data showing the dynein ATPase activity induced by X/XO or H2O2. (B) Representative Western blot gel document and summarized intensity ratio of Nox1 to b-actin showing the expression of Nox1 in CAMs with scramble (Scr) or Nox1 cDNA plasmid transfection. (C) Summarized ESR data showing the relative O2 . production in CAMs with scramble or Nox1 cDNA transfection in the absence or presence of DPI (50 lM). (D) Summarized data showing the effects of dynein inhib- itor EHNA (30 lM) on dynein ATPase activity in CAMs with scramble or Nox1 cDNA transfection. (E and F) Representative Western blot gel doc- ument and summarized data showing the protein expression of dynein in CAMs under X/XO or H2O2 stimulation (E) and after Nox1 cDNA transfection (F; n = 6 for all panels). *P < 0.05 versus Ctrl or Scram- ble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 1 Regulation of dynein ATPase activity by ROS. (A) CAMs were incubated with X/XO (10 lM/0.1 U/ml) or H2O2 (10 lM) for 24 hrs in the presence or absence of dynein inhibitor EHNA (30 lM). Summa- rized data showing the dynein ATPase activity induced by X/XO or H2O2. (B) Representative Western blot gel document and summarized intensity ratio of Nox1 to b-actin showing the expression of Nox1 in CAMs with scramble (Scr) or Nox1 cDNA plasmid transfection. (C) Summarized ESR data showing the relative O2 . production in CAMs with scramble or Nox1 cDNA transfection in the absence or presence of DPI (50 lM). (D) Summarized data showing the effects of dynein inhib- itor EHNA (30 lM) on dynein ATPase activity in CAMs with scramble or Nox1 cDNA transfection. (E and F) Representative Western blot gel doc- ument and summarized data showing the protein expression of dynein in CAMs under X/XO or H2O2 stimulation (E) and after Nox1 cDNA transfection (F; n = 6 for all panels). *P < 0.05 versus Ctrl or Scram- ble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Article Snippet: Nucleofection of DCTN2 cDNA and Nox1 cDNA Both dynamitin (Dynactin 2: DCTN2) cDNA (Catalog no. MC200162) and Nox1cDNA plasmids (Catalog no. MG226022) were purchased from OriGene Technologies.

Techniques: Activity Assay, Incubation, Western Blot, Expressing, Plasmid Preparation, Transfection, Inhibition

Fig. 2 Analysis of AP dynamic movements. (A) Typical ﬂuorescent images of LC3B-GFP-labelled AP in CAMs were taken every 10 sec. in CAMs. (B and C) The summarized data show the velocity of AP in CAMs pre-treated with EHNA (30 lM) or transfected with dynam- itin (DCTN2) cDNA (n = 6 for all panels). *P < 0.05 versus Scram- ble; #P < 0.05 versus CAMs with X/XO or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 2 Analysis of AP dynamic movements. (A) Typical ﬂuorescent images of LC3B-GFP-labelled AP in CAMs were taken every 10 sec. in CAMs. (B and C) The summarized data show the velocity of AP in CAMs pre-treated with EHNA (30 lM) or transfected with dynam- itin (DCTN2) cDNA (n = 6 for all panels). *P < 0.05 versus Scram- ble; #P < 0.05 versus CAMs with X/XO or Nox1 cDNA transfection alone.

Techniques: Transfection

Fig. 3 Inhibition of dynein activity blocked lysosome fusion. (A) Repre- sentative confocal microscopic images showing the co-localization of LC3B-GFP with Lamp1-RFP in live CAMs. (B) Summarized co-localiza- tion coefﬁcient (PCC) of LC3B and Lamp1 in CAMs transfected with scramble or DCTN2 cDNA. (C) Summarized co-localization coefﬁcient of LC3B-GFP and Lamp1-RFP in CAMs with or without EHNA (30 lM; n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 ver- sus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 3 Inhibition of dynein activity blocked lysosome fusion. (A) Repre- sentative confocal microscopic images showing the co-localization of LC3B-GFP with Lamp1-RFP in live CAMs. (B) Summarized co-localiza- tion coefﬁcient (PCC) of LC3B and Lamp1 in CAMs transfected with scramble or DCTN2 cDNA. (C) Summarized co-localization coefﬁcient of LC3B-GFP and Lamp1-RFP in CAMs with or without EHNA (30 lM; n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 ver- sus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Techniques: Inhibition, Activity Assay, Transfection

Fig. 4 Inhibition of dynein activity decreased APLs formation. Mouse CAMs were stained with acridine orange for 17 min. Representative dot plots of ﬂow cytometry (A) and summarized red-to-green ﬂuorescence ratio analysis showing APLs formation in CAMs with DCTN2 cDNA (B) or EHNA (C). (D) Summarized red-to-green ﬂuorescence ratio analysis showing APLs formation in CAMs with spautin-1 (10 lM) and leupeptin (0.25 mM). (E) Representative Western blot documents showing the expression of Lamp-1 from CAMs. (F) CAMs were treated with chloro- quine (CQ, 100 lM) for 30 min. or left untreated. They were then stained with LysoSensor Green DND-189 and analysed by ﬂuorescence microscopy (n = 6 for all panels). *P < 0.05 versus Scramble; #P < 0.05 versus CAMs with X/XO or H2O2 or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 4 Inhibition of dynein activity decreased APLs formation. Mouse CAMs were stained with acridine orange for 17 min. Representative dot plots of ﬂow cytometry (A) and summarized red-to-green ﬂuorescence ratio analysis showing APLs formation in CAMs with DCTN2 cDNA (B) or EHNA (C). (D) Summarized red-to-green ﬂuorescence ratio analysis showing APLs formation in CAMs with spautin-1 (10 lM) and leupeptin (0.25 mM). (E) Representative Western blot documents showing the expression of Lamp-1 from CAMs. (F) CAMs were treated with chloro- quine (CQ, 100 lM) for 30 min. or left untreated. They were then stained with LysoSensor Green DND-189 and analysed by ﬂuorescence microscopy (n = 6 for all panels). *P < 0.05 versus Scramble; #P < 0.05 versus CAMs with X/XO or H2O2 or Nox1 cDNA transfection alone.

Techniques: Inhibition, Activity Assay, Staining, Cytometry, Western Blot, Expressing, Microscopy, Transfection

Fig. 5 Dynein inhibition increased accumulation of AP and decreased breakdown of AP. (A and B) Summarized per cent of Cyto-ID-stained cells showing the relative number of AP in CAMs with EHNA or DCTN2 cDNA. (C and D) Representative confocal images and summarized co- localization coefﬁcient showing the co-localization of ubiquitin with p62 (n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 5 Dynein inhibition increased accumulation of AP and decreased breakdown of AP. (A and B) Summarized per cent of Cyto-ID-stained cells showing the relative number of AP in CAMs with EHNA or DCTN2 cDNA. (C and D) Representative confocal images and summarized co- localization coefﬁcient showing the co-localization of ubiquitin with p62 (n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Techniques: Inhibition, Staining, Ubiquitin Proteomics, Transfection

Fig. 7 Confocal microscopic detection of Ca2+ release from lysosomes locally in CAMs. (A) Representative confocal microscopy images show- ing Ca2+ release regions that co-localized with lysosomes as shown by yellow spots formed by green ﬂuo-4 signals with rhodamine-red lyso- somal marker (Lyso/Rho). Summarized data showing the co-localization coefﬁcient of Ca2+/ﬂuo-4 with Lyso/Rho signals in CAMs treated with X/XO or H2O2 (B) or Nox1 cDNA (C; n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 7 Confocal microscopic detection of Ca2+ release from lysosomes locally in CAMs. (A) Representative confocal microscopy images show- ing Ca2+ release regions that co-localized with lysosomes as shown by yellow spots formed by green ﬂuo-4 signals with rhodamine-red lyso- somal marker (Lyso/Rho). Summarized data showing the co-localization coefﬁcient of Ca2+/ﬂuo-4 with Lyso/Rho signals in CAMs treated with X/XO or H2O2 (B) or Nox1 cDNA (C; n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Techniques: Confocal Microscopy, Marker, Transfection

Fig. 8 Lysosome Ca2+ signalling-regulated dynein ATPase activity. (A) Ten microgram proteins of isolated cytoplasmic lysates from CAMs was incubated with X/XO (10 lM/0.1 U/ml) or H2O2 (10 lM) for 2 hrs. Summarized data showing the direct oxidizing effects of ROS on dynein ATPase activity. (B and C) Summarized data showing the effects of NA- ADP antagonists, PPADS (50 lM) or NED-19 (10 lM) on dynein ATPase activity in CAMs under X/XO (10 lM/0.1 U/ml) or H2O2 (10 lM) stimulation or Nox1 cDNA transfection in CAMs (n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 8 Lysosome Ca2+ signalling-regulated dynein ATPase activity. (A) Ten microgram proteins of isolated cytoplasmic lysates from CAMs was incubated with X/XO (10 lM/0.1 U/ml) or H2O2 (10 lM) for 2 hrs. Summarized data showing the direct oxidizing effects of ROS on dynein ATPase activity. (B and C) Summarized data showing the effects of NA- ADP antagonists, PPADS (50 lM) or NED-19 (10 lM) on dynein ATPase activity in CAMs under X/XO (10 lM/0.1 U/ml) or H2O2 (10 lM) stimulation or Nox1 cDNA transfection in CAMs (n = 6 for all panels). *P < 0.05 versus Ctrl or Scramble; #P < 0.05 versus CAMs treated with X/XO or H2O2 or Nox1 cDNA transfection alone.

Techniques: Activity Assay, Isolation, Incubation, Transfection

Fig. 9 NAADP-lysosome Ca2+-controlled high glucose–induced dynein activation. (A) Summarized data showing the effects of Nox1 inhibitor ML117 (100 lM) on O2 production in CAMs under control and high glucose (30 mM) for 48 hrs. Summarized data showing the effects of Nox1 inhibitor ML117, dynein inhibitor EHNA and NAADP antagonists PPADS or NED-19 on dynein ATPase activity (B) or APLs formation (red-to-green ﬂuorescence ratio of acridine orange; C) in CAMs under resting control condition or with high glucose (n = 6 for all panels). *P < 0.05 versus Ctrl; #P < 0.05 versus CAMs treated with high glu- cose.

Journal: Journal of cellular and molecular medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes.

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Fig. 9 NAADP-lysosome Ca2+-controlled high glucose–induced dynein activation. (A) Summarized data showing the effects of Nox1 inhibitor ML117 (100 lM) on O2 production in CAMs under control and high glucose (30 mM) for 48 hrs. Summarized data showing the effects of Nox1 inhibitor ML117, dynein inhibitor EHNA and NAADP antagonists PPADS or NED-19 on dynein ATPase activity (B) or APLs formation (red-to-green ﬂuorescence ratio of acridine orange; C) in CAMs under resting control condition or with high glucose (n = 6 for all panels). *P < 0.05 versus Ctrl; #P < 0.05 versus CAMs treated with high glu- cose.

Techniques: Activation Assay, Control, Activity Assay

Analysis of AP dynamic movements. ( A ) Typical fluorescent images of LC3B-GFP-labelled AP in CAMs were taken every 10 sec. in CAMs. ( B and C ) The summarized data show the velocity of AP in CAMs pre-treated with EHNA (30 μM) or transfected with dynamitin (DCTN2) cDNA ( n = 6 for all panels). * P < 0.05 versus Scramble; # P < 0.05 versus CAMs with X/XO or Nox1 cDNA transfection alone.

Journal: Journal of Cellular and Molecular Medicine

Article Title: Enhancement of dynein-mediated autophagosome trafficking and autophagy maturation by ROS in mouse coronary arterial myocytes

doi: 10.1111/jcmm.12326

Figure Lengend Snippet: Analysis of AP dynamic movements. ( A ) Typical fluorescent images of LC3B-GFP-labelled AP in CAMs were taken every 10 sec. in CAMs. ( B and C ) The summarized data show the velocity of AP in CAMs pre-treated with EHNA (30 μM) or transfected with dynamitin (DCTN2) cDNA ( n = 6 for all panels). * P < 0.05 versus Scramble; # P < 0.05 versus CAMs with X/XO or Nox1 cDNA transfection alone.

Article Snippet: Both dynamitin (Dynactin 2: DCTN2) cDNA (Catalog no. MC200162) and Nox1cDNA plasmids (Catalog no. MG226022) were purchased from OriGene Technologies.

Techniques: Transfection

Review

dctn2 cdna (OriGene)

Structured Review

Images

Related Articles

other:

Similar Products

Image Search Results