monoclonal anti cx43  (Cell Signaling Technology Inc)

Journal: Pharmacology Research & Perspectives

Article Title: β2‐Adrenergic Receptor Agonist Clenbuterol Protects Against Acute Ischemia/Reperfusion‐Induced Arrhythmia by Regulation of Akt/ eNOS / NO /Cx43 Signaling Pathway

doi: 10.1002/prp2.70070

Figure Lengend Snippet: Role of Cx43 in the anti‐arrhythmic effect of clenbuterol in anesthetized rats with acute ischemia/reperfusion arrhythmias. Ventricular arrhythmias were induced by ligating the left anterior descending (LAD) artery for 10 min, followed by a 10 min reperfusion. Clenbuterol (Clenb, 0.5 mg/kg, ip ) was administrated 1 h prior to ischemia; carbenoxolone (CBX, 10 mg/kg, iv ) was administrated 1 h before injection of clenbuterol. Representative immunoblots (A), and densitometric quantitative analysis of p‐Cx43/t‐Cx43 and np‐Cx43 (C) in rat ventricular tissue. ( n = 4, *** p < 0.001). Arrhythmia score (D), duration of VT + VF (E), and incidence of VP, VT, or VF (F) were recorded. ( n = 10–15, * p < 0.05, ns, no significance).

Article Snippet: The following primary antibodies: anti‐eNOS (1:500, #9572), anti‐p‐eNOS (1:1000, #9571S), anti‐Akt (1:000, #9272), anti‐p‐Akt (1:1000, #4060S) were purchased from Cell Signaling. anti‐GAPDH (1:5000, #abs830030) was purchased from Absin; a polyclonal anti‐Cx43 (1:2000, #71–0700) and a monoclonal anti‐Cx43 (1:1000, #13–8300) were purchased from Invitrogen.

Techniques: Injection, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: β2‐Adrenergic Receptor Agonist Clenbuterol Protects Against Acute Ischemia/Reperfusion‐Induced Arrhythmia by Regulation of Akt/ eNOS / NO /Cx43 Signaling Pathway

doi: 10.1002/prp2.70070

Figure Lengend Snippet: Role of β2‐adrenergic receptor in the anti‐arrhythmic effect of clenbuterol in anesthetized rats with acute ischemia/reperfusion arrhythmias. Ventricular arrhythmias were induced by ligating the left anterior descending (LAD) artery for 10 min, followed by a 10 min reperfusion. Clenbuterol (Clenb, 0.5 mg/kg, ip ) was administrated 1 h prior to ischemia; ICI 118551 (ICI, 5 mg/kg, ip ), metoprolol (Meto, 1 mg/kg, ip ) or propranolol (Prop, 10 mg/kg, ip ) was administrated 1 h before injection of clenbuterol. Arrhythmia score (A), duration of VT + VF (B), and incidence of VP, VT, or VF (C) were recorded. ( n = 10–15, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance). Representative immunoblots (D), and densitometric quantitative analysis (E) of p‐Cx43/t‐Cx43 and np‐Cx43 in rat ventricular tissue ( n = 3, * p < 0.05, *** p < 0.001).

Article Snippet: The following primary antibodies: anti‐eNOS (1:500, #9572), anti‐p‐eNOS (1:1000, #9571S), anti‐Akt (1:000, #9272), anti‐p‐Akt (1:1000, #4060S) were purchased from Cell Signaling. anti‐GAPDH (1:5000, #abs830030) was purchased from Absin; a polyclonal anti‐Cx43 (1:2000, #71–0700) and a monoclonal anti‐Cx43 (1:1000, #13–8300) were purchased from Invitrogen.

Techniques: Injection, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: β2‐Adrenergic Receptor Agonist Clenbuterol Protects Against Acute Ischemia/Reperfusion‐Induced Arrhythmia by Regulation of Akt/ eNOS / NO /Cx43 Signaling Pathway

doi: 10.1002/prp2.70070

Figure Lengend Snippet: Effect of eNOS on Cx43 dephosphorylation and effect of clenbuterol on anti‐arrhythmic effect of clenbuterol in anesthetized rats with acute ischemia/reperfusion arrhythmias. (A, B) Ventricular arrhythmias were induced by ligating the left anterior descending (LAD) artery for 10 min, followed by a 10 min reperfusion. Clenbuterol (Clenb, 0.5 mg/kg, ip ) was administrated 1 h prior to ischemia; ICI 118551 (ICI, 5 mg/kg, ip ) was administrated 1 h before injection of clenbuterol. Representative immunoblots (A), and densitometric quantitative analysis (B) of eNOS and phosphorylation of eNOS (p‐eNOS) in rat ventricular tissue ( n = 3, ** p < 0.01, *** p < 0.001). (C–I) Isolated mice hearts were subjected to 10 min global ischemia and 10 min reperfusion on a Langendorff apparatus. Clenbuterol (10 μmol/L) was administrated 10 min before global ischemia and through reperfusion (10 min); ICI 118551(1 μmol/L) or L‐NAME (100 μmol/L) pretreated the hearts for 10 min before clenbuterol treatment. (C) Arrhythmia score was recorded in indicated mice hearts. ( n = 8–10, *** p < 0.001). (D‐E) Representative immunoblots (D), and densitometric quantitative analysis (E) of eNOS and phosphorylation of eNOS (p‐eNOS) in mice ventricular tissue ( n = 3, *** p < 0.001). (F) NO concentrations were measured in indicated mice hearts. ( n = 3, * p < 0.05, *** p < 0.001). (G–I) Representative immunoblots (G), and densitometric quantitative analysis of p‐Cx43/t‐Cx43 (H) and expression of np‐Cx43 (I) in mice ventricular tissue ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001). (J) The distribution of Cx43 and co‐staining of wheat germ agglutinin (WGA) labeling were recorded by immunofluorescence microscopy, Cx43 located at intercalated disks (white arrows) and Cx43 located at lateral edges of the myocytes (yellow arrows) ( n = 4, Scale bar = 10 μm).

Article Snippet: The following primary antibodies: anti‐eNOS (1:500, #9572), anti‐p‐eNOS (1:1000, #9571S), anti‐Akt (1:000, #9272), anti‐p‐Akt (1:1000, #4060S) were purchased from Cell Signaling. anti‐GAPDH (1:5000, #abs830030) was purchased from Absin; a polyclonal anti‐Cx43 (1:2000, #71–0700) and a monoclonal anti‐Cx43 (1:1000, #13–8300) were purchased from Invitrogen.

Techniques: De-Phosphorylation Assay, Injection, Western Blot, Isolation, Expressing, Staining, Labeling, Immunofluorescence, Microscopy

Journal: Pharmacology Research & Perspectives

Article Title: β2‐Adrenergic Receptor Agonist Clenbuterol Protects Against Acute Ischemia/Reperfusion‐Induced Arrhythmia by Regulation of Akt/ eNOS / NO /Cx43 Signaling Pathway

doi: 10.1002/prp2.70070

Figure Lengend Snippet: Role of Akt/eNOS/Cx43 signaling pathway in the protection effect of clenbuterol on arrhythmia induced by acute ischemia/reperfusion. (A, B) Ventricular arrhythmias were induced by ligating the left anterior descending (LAD) artery for 10 min, followed by a 10 min reperfusion. Clenbuterol (Clenb, 0.5 mg/kg, ip ) was administrated 1 h prior to ischemia; ICI 118551 (ICI, 5 mg/kg, ip ) was administrated 1 h before injection of clenbuterol. Representative immunoblots (A), and densitometric quantitative analysis (B) of Akt and phosphorylation of Akt (p‐Akt) in rat ventricular tissue ( n = 3, *** p < 0.001). (C–I) Isolated mice hearts were subjected to 10 min global ischemia and 10 min reperfusion on a Langendorff apparatus. Clenbuterol (10 μmol/L) was administrated 10 min before global ischemia and through reperfusion (10 min); MK2206 (5 μmol/L) was administrated for 10 min before clenbuterol treatment. (C) Arrhythmia score was recorded in indicated hearts. ( n = 8–10, *** p < 0.001). (D–H) Representative immunoblots (D), and densitometric quantitative analysis (E‐H) of p‐Akt/Akt, p‐eNOS/eNOS, p‐Cx43/t‐Cx43, and np‐Cx43/GAPDH in mouse ventricular tissues ( n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001). (I) NO concentrations were measured in indicated hearts. ( n = 4, * p < 0.05, *** p < 0.001).

Article Snippet: The following primary antibodies: anti‐eNOS (1:500, #9572), anti‐p‐eNOS (1:1000, #9571S), anti‐Akt (1:000, #9272), anti‐p‐Akt (1:1000, #4060S) were purchased from Cell Signaling. anti‐GAPDH (1:5000, #abs830030) was purchased from Absin; a polyclonal anti‐Cx43 (1:2000, #71–0700) and a monoclonal anti‐Cx43 (1:1000, #13–8300) were purchased from Invitrogen.

Techniques: Injection, Western Blot, Isolation

Journal: Pharmacology Research & Perspectives

Article Title: β2‐Adrenergic Receptor Agonist Clenbuterol Protects Against Acute Ischemia/Reperfusion‐Induced Arrhythmia by Regulation of Akt/ eNOS / NO /Cx43 Signaling Pathway

doi: 10.1002/prp2.70070

Figure Lengend Snippet: Hypothetical scheme about the role of clenbuterol in cardiac ischemia/reperfusion‐induced arrhythmia. Acute ischemia/reperfusion injury increase non‐phosphorylated Cx43, promotes Cx43 lateralization, and consequently creates a highly arrhythmogenic substrate during early reperfusion. Clenbuterol activates the Akt/eNOS signaling pathway, augments NO bioavailability, maintains Cx43 phosphorylation, ultimately improving arrhythmias induced by ischemia/reperfusion injury.

Article Snippet: The following primary antibodies: anti‐eNOS (1:500, #9572), anti‐p‐eNOS (1:1000, #9571S), anti‐Akt (1:000, #9272), anti‐p‐Akt (1:1000, #4060S) were purchased from Cell Signaling. anti‐GAPDH (1:5000, #abs830030) was purchased from Absin; a polyclonal anti‐Cx43 (1:2000, #71–0700) and a monoclonal anti‐Cx43 (1:1000, #13–8300) were purchased from Invitrogen.

Techniques:

Journal: Investigative ophthalmology & visual science

Article Title: De-Differentiation of Corneal Epithelial Cells Into Functional Limbal Epithelial Stem Cells After the Ablation of Innate Stem Cells.

doi: 10.1167/iovs.65.13.32

Figure Lengend Snippet: FIGURE 2. The regeneration of LESCs at the limbus after innate stem cells ablation. (A) Immunostaining of potential LESCs markers (CK14, deltaN-p63, p75NTR, CD63, TSPAN7, IFITM3, ATF3, and ApoE), Cx43 and CECs marker CK12 in frozen sections of normal cornea. (B-D) Immunostaining of Cx43, ApoE, and CK12 in frozen sections of normal cornea and LESCs-ablation cornea at indicated days after the limbal epithelial removal. The limbus was shown. (E) The percentage of ApoE+ cells at the limbus, and MFI (limbal basal cells)/MFI (peripheral basal cells) of Cx43 and CK12 were determined to indicate the recovery of LESCs. N, normal cornea. Scale bars = 50 μm. Note that the limbus is localized based on the location of iris and ciliary body in the bright field and the expressions of LESC markers.

Article Snippet: The following primary antibodies were used: rabbit anti-CK12 monoclonal antibody (Abcam, ab185627; 1:400), rabbit anti-CK7 monoclonal antibody (Abcam, ab181598; 1:400), rabbit anti-connexin 43 (Cx43) monoclonal antibody (CST, #3512; 1:80), rabbit anti-ApoE monoclonal antibody (Abcam, ab183596; 1:400), rabbit anti-CK14 monoclonal antibody (Abcam, ab119695; 1:200), rabbit anti-deltaN-p63 polyclonal antibody (BioLegend, 619002; 1:400), rabbit anti-p75NTR monoclonal antibody (CST, #8238; 1:800), rabbit anti-CD63 polyclonal antibody (Bioworld, BS72936; 1:400), rabbit anti-TSPAN7 polyclonal antibody (Proteintech, 18695-1-AP; 1:100), rabbit anti-IFITM3 monoclonal antibody (CST, #59212; 1:100), mouse anti-ATF3 monoclonal antibody (Santa Cruz, sc-518032; 1:100), rabbit anti-YAP monoclonal antibody (CST, #14074; 1:100), rabbit anti-active-YAP (non-phosphorylated YAP) monoclonal antibody (Abcam, ab205270; 1:200), rabbit anti-Ki67 monoclonal antibody (CST, #9129; 1:100), rabbit anti-p-Histone H3-Ser10 (pH3) monoclonal antibody (CST, #53348; 1:100), and mouse antiCK15 monoclonal antibody (Santa Cruz, sc-47697; 1:200).

Techniques: Immunostaining, Marker

Journal: Investigative ophthalmology & visual science

Article Title: De-Differentiation of Corneal Epithelial Cells Into Functional Limbal Epithelial Stem Cells After the Ablation of Innate Stem Cells.

doi: 10.1167/iovs.65.13.32

Figure Lengend Snippet: FIGURE 6. The de-differentiation of CECs required an intact limbal niche. (A) Experimental strategy of the localized NaOH application to destroy the limbal niche, which allows the examination of the fate of CECs either with or without an intact limbal niche on the same cornea. (B) The whole-mount CK7 and EdU immunostaining against corneas of scratched limbus either with or without NaOH application. Dashed circles indicate the limbus. (C) The frozen-section CK7 and CK12 immunostaining against cornea of scratched limbus either with or without NaOH application at 10 and 20 days. (D–I) The frozen-section Cx43 and ApoE immunostaining against cornea of scratched limbus either with or without NaOH application at 6 and 10 days. The percentage of ApoE+ LESCs and MFI of Cx43 were quantified. Data are the mean ± SD, n = 5 biological replicates; limbus (S) = 35.59 ± 5.64, central cornea = 0 ± 0, and limbus (S+NaOH) = 0 ± 0 (E); limbus (S) = 17.05 ± 2.68, central cornea = 35.77 ± 7.27, and limbus (S+NaOH) = 48.26 ± 11.02 (F); limbus (S) = 51.33 ± 8.59, central cornea = 0.95 ± 2.13, and limbus (S+NaOH) = 3.12 ± 2.93 (H); limbus (S) = 29.17 ± 4.66, central cornea = 60.12 ± 24.45, and limbus (S+NaOH) = 55.05 ± 10.25 (I); statistical analysis were performed by paired 1-way ANOVA with Tukey’s test. Scale bars = 20 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-CK12 monoclonal antibody (Abcam, ab185627; 1:400), rabbit anti-CK7 monoclonal antibody (Abcam, ab181598; 1:400), rabbit anti-connexin 43 (Cx43) monoclonal antibody (CST, #3512; 1:80), rabbit anti-ApoE monoclonal antibody (Abcam, ab183596; 1:400), rabbit anti-CK14 monoclonal antibody (Abcam, ab119695; 1:200), rabbit anti-deltaN-p63 polyclonal antibody (BioLegend, 619002; 1:400), rabbit anti-p75NTR monoclonal antibody (CST, #8238; 1:800), rabbit anti-CD63 polyclonal antibody (Bioworld, BS72936; 1:400), rabbit anti-TSPAN7 polyclonal antibody (Proteintech, 18695-1-AP; 1:100), rabbit anti-IFITM3 monoclonal antibody (CST, #59212; 1:100), mouse anti-ATF3 monoclonal antibody (Santa Cruz, sc-518032; 1:100), rabbit anti-YAP monoclonal antibody (CST, #14074; 1:100), rabbit anti-active-YAP (non-phosphorylated YAP) monoclonal antibody (Abcam, ab205270; 1:200), rabbit anti-Ki67 monoclonal antibody (CST, #9129; 1:100), rabbit anti-p-Histone H3-Ser10 (pH3) monoclonal antibody (CST, #53348; 1:100), and mouse antiCK15 monoclonal antibody (Santa Cruz, sc-47697; 1:200).

Techniques: Immunostaining

Journal: Investigative ophthalmology & visual science

Article Title: De-Differentiation of Corneal Epithelial Cells Into Functional Limbal Epithelial Stem Cells After the Ablation of Innate Stem Cells.

doi: 10.1167/iovs.65.13.32

Figure Lengend Snippet: FIGURE 7. The role of Hippo/YAP pathway in the de-differentiation of CECs in vivo. (A) Immunostaining of YAP in frozen sections of normal cornea and LESCs-ablation cornea at indicated days after the limbal epithelial removal. The limbus was shown. (B, C) MFI and N/C ratio of YAP in limbal basal cells were quantified after the limbal epithelial removal. (D–F) The administration of vehicle or VTP or TRULI on cornea for 4 days after the LESCs ablation, and expressions of ApoE and Cx43 were examined by immunostaining at 6 days. The percentage of ApoE+ LESCs and MFI of Cx43 in limbal basal cells were quantified. (G–J) The frozen-section YAP, Ki67, and pH3 (indicators of cell proliferation) immunostaining against cornea of scratched limbus either with or without NaOH application at 10 days after the LESCs ablation. Arrows point to CECs with nuclear YAP. The MFI and N/C ratio of YAP in limbal basal cells and the percentage of Ki67+ limbal basal cells were quantified. (K–M) Corneas with scratched limbus and localized NaOH application were administrated with VTP for 4 days. The frozen-section ApoE and Cx43 immunostaining against cornea of scratched limbus either with or without NaOH application at 6 days. The percentage of ApoE+ LESCs and MFI of Cx43 in limbal basal cells were quantified. (N) Comparison of MFI of Cx43 in limbal basal cells

Article Snippet: The following primary antibodies were used: rabbit anti-CK12 monoclonal antibody (Abcam, ab185627; 1:400), rabbit anti-CK7 monoclonal antibody (Abcam, ab181598; 1:400), rabbit anti-connexin 43 (Cx43) monoclonal antibody (CST, #3512; 1:80), rabbit anti-ApoE monoclonal antibody (Abcam, ab183596; 1:400), rabbit anti-CK14 monoclonal antibody (Abcam, ab119695; 1:200), rabbit anti-deltaN-p63 polyclonal antibody (BioLegend, 619002; 1:400), rabbit anti-p75NTR monoclonal antibody (CST, #8238; 1:800), rabbit anti-CD63 polyclonal antibody (Bioworld, BS72936; 1:400), rabbit anti-TSPAN7 polyclonal antibody (Proteintech, 18695-1-AP; 1:100), rabbit anti-IFITM3 monoclonal antibody (CST, #59212; 1:100), mouse anti-ATF3 monoclonal antibody (Santa Cruz, sc-518032; 1:100), rabbit anti-YAP monoclonal antibody (CST, #14074; 1:100), rabbit anti-active-YAP (non-phosphorylated YAP) monoclonal antibody (Abcam, ab205270; 1:200), rabbit anti-Ki67 monoclonal antibody (CST, #9129; 1:100), rabbit anti-p-Histone H3-Ser10 (pH3) monoclonal antibody (CST, #53348; 1:100), and mouse antiCK15 monoclonal antibody (Santa Cruz, sc-47697; 1:200).

Techniques: In Vivo, Immunostaining, Comparison