phosphorylated iκb α ser32  (Cell Signaling Technology Inc)

Journal: PLoS ONE

Article Title: The Anti-Inflammatory Effects of Flavanol-Rich Lychee Fruit Extract in Rat Hepatocytes

doi: 10.1371/journal.pone.0093818

Figure Lengend Snippet: ( A ) FRLFE decreases the iNOS promoter activity. Hepatocytes were transfected with an iNOS promoter–luciferase construct (pRiNOS-Luc-3′UTR; top) and pCMV-LacZ (internal control) and were subsequently treated with IL-1β and/or FRLFE. κB, NF-κB-binding site; TATA, TATA box. The luciferase activity in the transfected cells was normalized to the β-galactosidase activity, and the fold activation was calculated by dividing the normalized luciferase activity by the luciferase activity in the presence of IL-1β alone. The data represent the mean ± SD ( n = 3). * P <0.05, ** P <0.01 versus IL-1β alone. ( B ) Nuclear extracts from FRLFE-treated hepatocytes decrease the DNA-binding activity to an NF-κB-binding site. Nuclear extracts were prepared from the cells and were analyzed using an EMSA to detect the NF-κB that was bound to a radiolabeled DNA probe harboring an NF-κB-binding site (κB). Competitor (Comp), cold DNA probe that was added to the reaction mixture at 100-fold molar excess to the radiolabeled probe. ( C ) FRLFE does not directly inhibit the DNA-binding activity of nuclear NF-κB. To induce NF-κB, hepatocytes were treated with IL-1β alone for 0.5 h, and a nuclear extract (NE) was prepared from these cells. The nuclear extract was directly mixed with FRLFE and analyzed using an EMSA to detect the NF-κB that was bound to the DNA probe harboring an NF-κB-binding site, similarly to (B). ( D ) FRLFE decreases the phosphorylation of IκB-α. Hepatocytes were treated with IL-1β and/or FRLFE for the indicated times. Hepatocyte extracts were immunoblotted with an anti-IκB-α, anti-phosphorylated IκB-α (p-IκB-α), or anti-β-tubulin antibody (internal control).

Article Snippet: Immunostaining was performed using primary antibodies that had been raised against rat iNOS (Thermo Fisher Scientific, Waltham, MA, USA), human NF-κB inhibitor α (IκB-α; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated IκB-α (Ser32/36 [5A5]), and rat β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), followed by visualization with the Enhanced Chemiluminescence Blotting Detection Reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: The Anti-Inflammatory Effects of Flavanol-Rich Lychee Fruit Extract in Rat Hepatocytes

doi: 10.1371/journal.pone.0093818

Figure Lengend Snippet: Transcripts reduced by FRLFE in rat hepatocytes.

Article Snippet: Immunostaining was performed using primary antibodies that had been raised against rat iNOS (Thermo Fisher Scientific, Waltham, MA, USA), human NF-κB inhibitor α (IκB-α; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated IκB-α (Ser32/36 [5A5]), and rat β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), followed by visualization with the Enhanced Chemiluminescence Blotting Detection Reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA).

Techniques:

Journal: PLoS ONE

Article Title: The Anti-Inflammatory Effects of Flavanol-Rich Lychee Fruit Extract in Rat Hepatocytes

doi: 10.1371/journal.pone.0093818

Figure Lengend Snippet: A pathway to activate the iNOS gene and the action of FRLFE are schematically depicted. The bold arrows indicate the decreases caused by FRLFE in this study. The proinflammatory cytokine IL-1β binds to its receptor (type I IL-1 receptor, IL1R1) to activate NF-κB through the IκB kinase (IKK) signaling pathway , . Activated IKK phosphorylates IκB-α, resulting in the degradation of IκB-α. A circled P denotes protein phosphorylation. Active NF-κB enters into the nucleus, binds to the iNOS gene promoter (κB sites), and activates transcription. FRLFE inhibits the phosphorylation of IκB-α and the nuclear translocation of NF-κB, resulting in a decrease in the nuclear levels of NF-κB. Because NF-κB also regulates the transcription of the iNOS asRNA, FRLFE reduces the level of iNOS asRNA, which interacts with and stabilizes the iNOS mRNA . Furthermore, FRLFE may interfere with the iNOS mRNA–asRNA interaction at a posttranscriptional level. Therefore, FRLFE significantly decreases the level of iNOS mRNA.

Article Snippet: Immunostaining was performed using primary antibodies that had been raised against rat iNOS (Thermo Fisher Scientific, Waltham, MA, USA), human NF-κB inhibitor α (IκB-α; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated IκB-α (Ser32/36 [5A5]), and rat β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), followed by visualization with the Enhanced Chemiluminescence Blotting Detection Reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA).

Techniques: Translocation Assay