Journal: Molecular Vision

Article Title: Epac1 agonist decreased inflammatory proteins in retinal endothelial cells, and loss of Epac1 increased inflammatory proteins in the retinal vasculature of mice

doi:

Figure Lengend Snippet: Epac1 agonist decreased TNF-α and IL-1β in vitro. Data show results from retinal endothelial cells (RECs) grown in normal glucose (NG) or high glucose (HG) only, grown in NG or HG and treated with CPT-2’-O-Me-cAMP, an exchange protein for cAMP 1 (Epac1)-specific agonist (NG or HG+8CPT-cAMP), and grown in NG and HG and treated with scrambled (NG or HG+Sc) or Epac1 siRNA (NG or HG+Epac1 siRNA). A : Western blot data show successful knockdown of Epac1. B : Western blot data for Epac2 show that CPT-2’-O-Me-cAMP is specific for Epac1. C : Enzyme-linked immunosorbent assay (ELISA) data for tumor necrosis factor alpha (TNF-α). D : Data for interleukin-1β (IL-1β). E : Western blot results for nuclear factor kappa beta (NFκB). F : Western blot results for inhibitor of kappa beta (IκB). n = 4 for each group. Data are mean ± standard error of the mean (SEM). * p<0.05 versus NG, #p<0.05 versus HG, $p<0.05 versus HG+8CPT-cAMP.

Article Snippet: After blocking in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and 5% (w/v) bovine serum albumin, the membranes were treated with Epac1 (ab109415), Epac 2 (ab193665, Abcam), total nuclear factor kappa beta (NFκB; #4764), phosphorylated NFκB (Ser 536, #3303), phosphorylated IκB (Ser32, #2859), total IκB (#4812, Cell Signaling, Danvers, MA), and beta actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase.

Techniques: In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Epac1 agonist decreased inflammatory proteins in retinal endothelial cells, and loss of Epac1 increased inflammatory proteins in the retinal vasculature of mice

doi:

Figure Lengend Snippet: Epac1 regulates NFκB phosphorylation in the mouse retina. Whole retinal lysates from exchange protein for cAMP 1 (Epac1) floxed versus Epac1 Cre-Lox mice were processed for western blot analyses for phosphorylated nuclear factor kappa beta (NFκB (Ser536) versus total NFκB ( A ) or inhibitor of kappa beta (IκB; B ). n = 5 for each group. Data are mean ± standard error of the mean (SEM). *p<0.05 versus Epac1 floxed.

Article Snippet: After blocking in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and 5% (w/v) bovine serum albumin, the membranes were treated with Epac1 (ab109415), Epac 2 (ab193665, Abcam), total nuclear factor kappa beta (NFκB; #4764), phosphorylated NFκB (Ser 536, #3303), phosphorylated IκB (Ser32, #2859), total IκB (#4812, Cell Signaling, Danvers, MA), and beta actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase.

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: JNK-IN-8 treatment alleviates lipopolysaccharide-induced acute lung injury via suppression of inflammation and oxidative stress regulated by JNK/NF-κB signaling

doi: 10.3892/mmr.2020.11789

Figure Lengend Snippet: JNK-IN-8 suppresses the JNK/NF-κB signaling pathway in LPS-induced acute lung injury in vivo and in vitro . (A) Primary macrophages stimulated by LPS and JNK-IN-8 (10 mM) for 2 h were extracted and subjected to western blot analysis. The protein expression levels of JNK, p-JNK, p65 and p-p65 were assessed and (B) analyzed by densitometry. (C) Primary macrophages, stimulated with LPS in the presence or absence of JNK-IN-8 (10 mM) for 0, 30, 60 or 120 min, were extracted and subjected to western blot analysis. The protein expression levels of JNK, p-JNK, p65 and p-p65 were assessed and (D) analyzed by densitometry. (E) Protein expression levels of JNK, p-JNK, p65 and p-p65 in lung tissue were assessed and (F) analyzed by densitometry. β-actin was used as the internal loading control. (G) Primary macrophages were stimulated with LPS in the presence or absence of JNK-IN-8 (10 mM) for 2 h. Cytoplasmic and nuclear NF-κB p65 protein expression levels were determined and (H) analyzed by densitometry. β-actin and Lamin B were used as internal loading controls. Data are presented as the mean ± SD (n=3). ***P<0.001 vs. Sham; ## P<0.01 and ### P<0.001 vs. LPS. LPS, lipopolysaccharide; p-, phosphorylated.

Article Snippet: Specific primary antibodies against NF-κB p65 (cat. no. 8242), phosphorylated (p)-p65 (Ser536; cat. no. 3303), JNK (cat. no. 9252), p-JNK (Thr183/Tyr185; cat. no. 4668) and β-actin (cat. no. 3700) were purchased from Cell Signaling Technology, Inc.

Techniques: In Vivo, In Vitro, Western Blot, Expressing