rabbit anti phospho cx43 ser373  (Thermo Fisher)


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    Name:
    Phospho Connexin 43 Ser373 Polyclonal Antibody
    Description:
    Phospho Connexin 43 Ser373 Polyclonal Antibody for Western Blot
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    pa564670
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    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher rabbit anti phospho cx43 ser373
    Schematic showing potential roles of astrocytic <t>Cx43,</t> hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of <t>Ser373-phosphorylated</t> Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity
    Phospho Connexin 43 Ser373 Polyclonal Antibody for Western Blot
    https://www.bioz.com/result/rabbit anti phospho cx43 ser373/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho cx43 ser373 - by Bioz Stars, 2020-08
    92/100 stars

    Images

    1) Product Images from "Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone"

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-018-1127-3

    Schematic showing potential roles of astrocytic Cx43, hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of Ser373-phosphorylated Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity
    Figure Legend Snippet: Schematic showing potential roles of astrocytic Cx43, hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of Ser373-phosphorylated Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity

    Techniques Used: Activation Assay, Activity Assay

    OGD/R injury with SalB or CBX application influenced astrocytic phosphorylated Cx43 and corresponding kinases. a1 , a2 OGD/R injury had no significant effect on cytoplasmic PKCε levels but significantly increased plasma membrane levels of PKCε and its Ser729-phosphorylated activated state. SalB and CBX reduced PKCε levels in the plasma membrane and increased them in the cytoplasm. Conversely, the OGD/R group’s Ser368-phosphorylated Cx43 levels were decreased in the plasma membrane and increased in the cytoplasm. SalB reversed these effects, but CBX reduced Ser368-phosphorylated Cx43 levels. b1 , b2 The OGD/R group exhibited decreased cytoplasmic levels of Thr308-phosphorylated PKB, though SalB reversed this effect. There were no significant changes in the plasma membrane levels. Furthermore, the OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Ser373-phosphorylated Cx43. SalB reduced the plasma membrane levels but further increased the cytoplasmic levels. CBX had little effect on the levels of Ser373-phosphorylated Cx43 and Thr308-phosphorylated PKB. c1 , c2 The OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. The OGD/R group also exhibited elevated plasma membrane levels of Src, which may be related to the elevated Tyr416-phosphorylated Src levels. SalB increased the plasma membrane levels of Src’s Tyr527-phosphorylated deactivated form but did not significantly affect plasma membrane levels of Tyr416-phosphorylated Src. CBX significantly reduced cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. Furthermore, the OGD/R group exhibited increased cytoplasmic and plasma membrane levels of Tyr265-phosphorylated Cx43. SalB reversed this effect, but CBX achieved only non-significant reduction of the plasma membrane levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. * p
    Figure Legend Snippet: OGD/R injury with SalB or CBX application influenced astrocytic phosphorylated Cx43 and corresponding kinases. a1 , a2 OGD/R injury had no significant effect on cytoplasmic PKCε levels but significantly increased plasma membrane levels of PKCε and its Ser729-phosphorylated activated state. SalB and CBX reduced PKCε levels in the plasma membrane and increased them in the cytoplasm. Conversely, the OGD/R group’s Ser368-phosphorylated Cx43 levels were decreased in the plasma membrane and increased in the cytoplasm. SalB reversed these effects, but CBX reduced Ser368-phosphorylated Cx43 levels. b1 , b2 The OGD/R group exhibited decreased cytoplasmic levels of Thr308-phosphorylated PKB, though SalB reversed this effect. There were no significant changes in the plasma membrane levels. Furthermore, the OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Ser373-phosphorylated Cx43. SalB reduced the plasma membrane levels but further increased the cytoplasmic levels. CBX had little effect on the levels of Ser373-phosphorylated Cx43 and Thr308-phosphorylated PKB. c1 , c2 The OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. The OGD/R group also exhibited elevated plasma membrane levels of Src, which may be related to the elevated Tyr416-phosphorylated Src levels. SalB increased the plasma membrane levels of Src’s Tyr527-phosphorylated deactivated form but did not significantly affect plasma membrane levels of Tyr416-phosphorylated Src. CBX significantly reduced cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. Furthermore, the OGD/R group exhibited increased cytoplasmic and plasma membrane levels of Tyr265-phosphorylated Cx43. SalB reversed this effect, but CBX achieved only non-significant reduction of the plasma membrane levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. * p

    Techniques Used:

    Related Articles

    Western Blot:

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone
    Article Snippet: .. Western blot analysis Protein concentration was evaluated using the BCA method, and 20–40 μg of total extract was separated on SDS polyacrilamide gels, and then the gel-separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the indicated primary antibodies: overnight at 4 °C with Rabbit anti-pan Cx43(1:1000, Cell signaling technology, Cat#3512); Rabbit anti-phospho-Cx43/ GJA1(Ser 368)(1:1000, Abcam,Cat#ab30559); Rabbit anti-phospho-Cx43 (Ser373) (1:1000, Thermo Fisher Scientific, Cat#PA5-64670); Rabbit anti-phospho-Cx43(Ser265)(1:500, Thermo Fisher Scientific, Cat#PA5-37584); Rabbit anti-PKCε (1:200, Santa Cruz Biotechnology, Cat#sc-214); Goat anti-phospho-PKCε (Ser729) (1: 200, Santa Cruz Biotechnology, Cat#sc-12355); Rabbit anti-Akt(PKB) (1:1000, Cell Signaling Technology, Cat#4691); Rabbit anti-phospho-Akt(Thr308) antibody (1:1000, Cell Signaling Technology, Cat#13038); Rabbit anti-Src (1:1000, Cell Signaling Technology, Cat#2108); Rabbit anti-Phospho-Src(Tyr527) (1:1000, Cell Signaling Technology, Cat#2105); Rabbit anti-Phospho-Src(Tyr416) (1:1000, Cell Signaling Technology, Cat#6943); Rabbit anti-GAPDH antibody (1/5000, Abcam, Cat#ab37168); mouse anti-β-actin(1:1000, Abcam, Cat#ab6276); Rabbit anti-Arginase-1 (1:1000, Cell Signaling Technology, Cat#93668). .. The membranes were washed and then incubated with secondary antibody for 1 h and then analyzed by an Odyssey infrared imaging system (LiCor, USA).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone
    Article Snippet: .. Western blot analysis Protein concentration was evaluated using the BCA method, and 20–40 μg of total extract was separated on SDS polyacrilamide gels, and then the gel-separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the indicated primary antibodies: overnight at 4 °C with Rabbit anti-pan Cx43(1:1000, Cell signaling technology, Cat3512); Rabbit anti-phospho-Cx43/ GJA1(Ser 368)(1:1000, Abcam,Cat#ab30559); Rabbit anti-phospho-Cx43 (Ser373) (1:1000, Thermo Fisher Scientific, Cat#PA5-64670); Rabbit anti-phospho-Cx43(Ser265)(1:500, Thermo Fisher Scientific, Cat#PA5-37584); Rabbit anti-PKCε (1:200, Santa Cruz Biotechnology, Cat#sc-214); Goat anti-phospho-PKCε (Ser729) (1: 200, Santa Cruz Biotechnology, Cat#sc-12355); Rabbit anti-Akt(PKB) (1:1000, Cell Signaling Technology, Cat#4691); Rabbit anti-phospho-Akt(Thr308) antibody (1:1000, Cell Signaling Technology, Cat#13038); Rabbit anti-Src (1:1000, Cell Signaling Technology, Cat#2108); Rabbit anti-Phospho-Src(Tyr527) (1:1000, Cell Signaling Technology, Cat#2105); Rabbit anti-Phospho-Src(Tyr416) (1:1000, Cell Signaling Technology, Cat#6943); Rabbit anti-GAPDH antibody (1/5000, Abcam, Cat#ab37168); mouse anti-β-actin(1:1000, Abcam, Cat#ab6276); Rabbit anti-Arginase-1 (1:1000, Cell Signaling Technology, Cat#93668). .. The membranes were washed and then incubated with secondary antibody for 1 h and then analyzed by an Odyssey infrared imaging system (LiCor, USA).

    Protein Concentration:

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone
    Article Snippet: .. Western blot analysis Protein concentration was evaluated using the BCA method, and 20–40 μg of total extract was separated on SDS polyacrilamide gels, and then the gel-separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the indicated primary antibodies: overnight at 4 °C with Rabbit anti-pan Cx43(1:1000, Cell signaling technology, Cat#3512); Rabbit anti-phospho-Cx43/ GJA1(Ser 368)(1:1000, Abcam,Cat#ab30559); Rabbit anti-phospho-Cx43 (Ser373) (1:1000, Thermo Fisher Scientific, Cat#PA5-64670); Rabbit anti-phospho-Cx43(Ser265)(1:500, Thermo Fisher Scientific, Cat#PA5-37584); Rabbit anti-PKCε (1:200, Santa Cruz Biotechnology, Cat#sc-214); Goat anti-phospho-PKCε (Ser729) (1: 200, Santa Cruz Biotechnology, Cat#sc-12355); Rabbit anti-Akt(PKB) (1:1000, Cell Signaling Technology, Cat#4691); Rabbit anti-phospho-Akt(Thr308) antibody (1:1000, Cell Signaling Technology, Cat#13038); Rabbit anti-Src (1:1000, Cell Signaling Technology, Cat#2108); Rabbit anti-Phospho-Src(Tyr527) (1:1000, Cell Signaling Technology, Cat#2105); Rabbit anti-Phospho-Src(Tyr416) (1:1000, Cell Signaling Technology, Cat#6943); Rabbit anti-GAPDH antibody (1/5000, Abcam, Cat#ab37168); mouse anti-β-actin(1:1000, Abcam, Cat#ab6276); Rabbit anti-Arginase-1 (1:1000, Cell Signaling Technology, Cat#93668). .. The membranes were washed and then incubated with secondary antibody for 1 h and then analyzed by an Odyssey infrared imaging system (LiCor, USA).

    BIA-KA:

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone
    Article Snippet: .. Western blot analysis Protein concentration was evaluated using the BCA method, and 20–40 μg of total extract was separated on SDS polyacrilamide gels, and then the gel-separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the indicated primary antibodies: overnight at 4 °C with Rabbit anti-pan Cx43(1:1000, Cell signaling technology, Cat#3512); Rabbit anti-phospho-Cx43/ GJA1(Ser 368)(1:1000, Abcam,Cat#ab30559); Rabbit anti-phospho-Cx43 (Ser373) (1:1000, Thermo Fisher Scientific, Cat#PA5-64670); Rabbit anti-phospho-Cx43(Ser265)(1:500, Thermo Fisher Scientific, Cat#PA5-37584); Rabbit anti-PKCε (1:200, Santa Cruz Biotechnology, Cat#sc-214); Goat anti-phospho-PKCε (Ser729) (1: 200, Santa Cruz Biotechnology, Cat#sc-12355); Rabbit anti-Akt(PKB) (1:1000, Cell Signaling Technology, Cat#4691); Rabbit anti-phospho-Akt(Thr308) antibody (1:1000, Cell Signaling Technology, Cat#13038); Rabbit anti-Src (1:1000, Cell Signaling Technology, Cat#2108); Rabbit anti-Phospho-Src(Tyr527) (1:1000, Cell Signaling Technology, Cat#2105); Rabbit anti-Phospho-Src(Tyr416) (1:1000, Cell Signaling Technology, Cat#6943); Rabbit anti-GAPDH antibody (1/5000, Abcam, Cat#ab37168); mouse anti-β-actin(1:1000, Abcam, Cat#ab6276); Rabbit anti-Arginase-1 (1:1000, Cell Signaling Technology, Cat#93668). .. The membranes were washed and then incubated with secondary antibody for 1 h and then analyzed by an Odyssey infrared imaging system (LiCor, USA).

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    Thermo Fisher rabbit anti phospho cx43 ser373
    Schematic showing potential roles of astrocytic <t>Cx43,</t> hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of <t>Ser373-phosphorylated</t> Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity
    Rabbit Anti Phospho Cx43 Ser373, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho cx43 ser373/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho cx43 ser373 - by Bioz Stars, 2020-08
    92/100 stars
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    Schematic showing potential roles of astrocytic Cx43, hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of Ser373-phosphorylated Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity

    Journal: Journal of Neuroinflammation

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

    doi: 10.1186/s12974-018-1127-3

    Figure Lengend Snippet: Schematic showing potential roles of astrocytic Cx43, hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of Ser373-phosphorylated Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity

    Article Snippet: Western blot analysis Protein concentration was evaluated using the BCA method, and 20–40 μg of total extract was separated on SDS polyacrilamide gels, and then the gel-separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the indicated primary antibodies: overnight at 4 °C with Rabbit anti-pan Cx43(1:1000, Cell signaling technology, Cat#3512); Rabbit anti-phospho-Cx43/ GJA1(Ser 368)(1:1000, Abcam,Cat#ab30559); Rabbit anti-phospho-Cx43 (Ser373) (1:1000, Thermo Fisher Scientific, Cat#PA5-64670); Rabbit anti-phospho-Cx43(Ser265)(1:500, Thermo Fisher Scientific, Cat#PA5-37584); Rabbit anti-PKCε (1:200, Santa Cruz Biotechnology, Cat#sc-214); Goat anti-phospho-PKCε (Ser729) (1: 200, Santa Cruz Biotechnology, Cat#sc-12355); Rabbit anti-Akt(PKB) (1:1000, Cell Signaling Technology, Cat#4691); Rabbit anti-phospho-Akt(Thr308) antibody (1:1000, Cell Signaling Technology, Cat#13038); Rabbit anti-Src (1:1000, Cell Signaling Technology, Cat#2108); Rabbit anti-Phospho-Src(Tyr527) (1:1000, Cell Signaling Technology, Cat#2105); Rabbit anti-Phospho-Src(Tyr416) (1:1000, Cell Signaling Technology, Cat#6943); Rabbit anti-GAPDH antibody (1/5000, Abcam, Cat#ab37168); mouse anti-β-actin(1:1000, Abcam, Cat#ab6276); Rabbit anti-Arginase-1 (1:1000, Cell Signaling Technology, Cat#93668).

    Techniques: Activation Assay, Activity Assay

    OGD/R injury with SalB or CBX application influenced astrocytic phosphorylated Cx43 and corresponding kinases. a1 , a2 OGD/R injury had no significant effect on cytoplasmic PKCε levels but significantly increased plasma membrane levels of PKCε and its Ser729-phosphorylated activated state. SalB and CBX reduced PKCε levels in the plasma membrane and increased them in the cytoplasm. Conversely, the OGD/R group’s Ser368-phosphorylated Cx43 levels were decreased in the plasma membrane and increased in the cytoplasm. SalB reversed these effects, but CBX reduced Ser368-phosphorylated Cx43 levels. b1 , b2 The OGD/R group exhibited decreased cytoplasmic levels of Thr308-phosphorylated PKB, though SalB reversed this effect. There were no significant changes in the plasma membrane levels. Furthermore, the OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Ser373-phosphorylated Cx43. SalB reduced the plasma membrane levels but further increased the cytoplasmic levels. CBX had little effect on the levels of Ser373-phosphorylated Cx43 and Thr308-phosphorylated PKB. c1 , c2 The OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. The OGD/R group also exhibited elevated plasma membrane levels of Src, which may be related to the elevated Tyr416-phosphorylated Src levels. SalB increased the plasma membrane levels of Src’s Tyr527-phosphorylated deactivated form but did not significantly affect plasma membrane levels of Tyr416-phosphorylated Src. CBX significantly reduced cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. Furthermore, the OGD/R group exhibited increased cytoplasmic and plasma membrane levels of Tyr265-phosphorylated Cx43. SalB reversed this effect, but CBX achieved only non-significant reduction of the plasma membrane levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. * p

    Journal: Journal of Neuroinflammation

    Article Title: Roles of astrocytic connexin-43, hemichannels, and gap junctions in oxygen-glucose deprivation/reperfusion injury induced neuroinflammation and the possible regulatory mechanisms of salvianolic acid B and carbenoxolone

    doi: 10.1186/s12974-018-1127-3

    Figure Lengend Snippet: OGD/R injury with SalB or CBX application influenced astrocytic phosphorylated Cx43 and corresponding kinases. a1 , a2 OGD/R injury had no significant effect on cytoplasmic PKCε levels but significantly increased plasma membrane levels of PKCε and its Ser729-phosphorylated activated state. SalB and CBX reduced PKCε levels in the plasma membrane and increased them in the cytoplasm. Conversely, the OGD/R group’s Ser368-phosphorylated Cx43 levels were decreased in the plasma membrane and increased in the cytoplasm. SalB reversed these effects, but CBX reduced Ser368-phosphorylated Cx43 levels. b1 , b2 The OGD/R group exhibited decreased cytoplasmic levels of Thr308-phosphorylated PKB, though SalB reversed this effect. There were no significant changes in the plasma membrane levels. Furthermore, the OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Ser373-phosphorylated Cx43. SalB reduced the plasma membrane levels but further increased the cytoplasmic levels. CBX had little effect on the levels of Ser373-phosphorylated Cx43 and Thr308-phosphorylated PKB. c1 , c2 The OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. The OGD/R group also exhibited elevated plasma membrane levels of Src, which may be related to the elevated Tyr416-phosphorylated Src levels. SalB increased the plasma membrane levels of Src’s Tyr527-phosphorylated deactivated form but did not significantly affect plasma membrane levels of Tyr416-phosphorylated Src. CBX significantly reduced cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. Furthermore, the OGD/R group exhibited increased cytoplasmic and plasma membrane levels of Tyr265-phosphorylated Cx43. SalB reversed this effect, but CBX achieved only non-significant reduction of the plasma membrane levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. * p

    Article Snippet: Western blot analysis Protein concentration was evaluated using the BCA method, and 20–40 μg of total extract was separated on SDS polyacrilamide gels, and then the gel-separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and probed using the indicated primary antibodies: overnight at 4 °C with Rabbit anti-pan Cx43(1:1000, Cell signaling technology, Cat#3512); Rabbit anti-phospho-Cx43/ GJA1(Ser 368)(1:1000, Abcam,Cat#ab30559); Rabbit anti-phospho-Cx43 (Ser373) (1:1000, Thermo Fisher Scientific, Cat#PA5-64670); Rabbit anti-phospho-Cx43(Ser265)(1:500, Thermo Fisher Scientific, Cat#PA5-37584); Rabbit anti-PKCε (1:200, Santa Cruz Biotechnology, Cat#sc-214); Goat anti-phospho-PKCε (Ser729) (1: 200, Santa Cruz Biotechnology, Cat#sc-12355); Rabbit anti-Akt(PKB) (1:1000, Cell Signaling Technology, Cat#4691); Rabbit anti-phospho-Akt(Thr308) antibody (1:1000, Cell Signaling Technology, Cat#13038); Rabbit anti-Src (1:1000, Cell Signaling Technology, Cat#2108); Rabbit anti-Phospho-Src(Tyr527) (1:1000, Cell Signaling Technology, Cat#2105); Rabbit anti-Phospho-Src(Tyr416) (1:1000, Cell Signaling Technology, Cat#6943); Rabbit anti-GAPDH antibody (1/5000, Abcam, Cat#ab37168); mouse anti-β-actin(1:1000, Abcam, Cat#ab6276); Rabbit anti-Arginase-1 (1:1000, Cell Signaling Technology, Cat#93668).

    Techniques: