Article Title: Mechanism of Ang II involvement in activation of NF-?B through phosphorylation of p65 during aging
Figure Lengend Snippet: Effect of Ang II on NF-κB activation in YPEN-1 cells. Cells were stimulated by Ang II and incubated from 5 to 30 min. Cells lysate were analyzed by Western blot analysis using IκBα-, anti-p-IκBα-, anti-p65-, and anti-p50-specific antibodies (a). Cells lysate were analyzed by Western blot analysis using p-IKKαβ- and p-p65 (Ser 536)-specific antibodies (b). The protein–protein interaction between IKKα and phosphorylated p65 was examined by immunoprecipitation. One representative blot is shown from three experiments that yielded similar results (c). Cells lysate were analyzed by Western blot analysis using p-ERK-, p-MSK-1-, and p-p65 (Ser 276)-specific antibodies (d). Immunocytochemistry was performed using p-p65 (Ser 276)-specific polyclonal antibody. Ser 276 phosphorylation of the p65 protein was labeled with Alexa 488 fluorescence (green). Ser 276 phosphorylation of translocated p65 could be identified by nuclear staining with 1 mg/mL of Hochest 33342 (blue) for 5 min (e). Cells were stimulated by Ang II and incubated from 30 min to 24 h. Cells lysate were analyzed by Western blot analysis using COX-2- and AT1-specific antibodies (f). Anti-β-actin and anti-TFIIB antibodies were used to evaluate equal protein loading. One representative blot is shown from three experiments that yielded similar results. Results of one-factor ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. AT1 angiotensin II type 1 receptor, ERK extracellular signal-regulated kinase, COX-2 cyclooxygenase-2, IKKαβ IκB kinase αβ, MSK mitogen- and stress-activated protein kinase 1, TFIIB transcription factor II B
Article Snippet: YPEN-1 cells were obtained from ATCC (Manassas, VA, USA).
Techniques: Activation Assay, Incubation, Western Blot, Immunoprecipitation, Immunocytochemistry, Labeling, Fluorescence, Staining