Journal: Age

Article Title: Mechanism of Ang II involvement in activation of NF-?B through phosphorylation of p65 during aging

doi: 10.1007/s11357-011-9207-7

Figure Lengend Snippet: Effect of Ang II on redox imbalances in YPEN-1 cells. Cells were treated with various doses of Ang II for 30 min. Intracellular RS levels were measured by DCF-DA using a fluorescent probe (Genious) (a). Intracellular RS levels were measured by DCF-DA using a fluorescent probe (Motic) control (a), 1 μM (b), 5 μM (c), 10 μM (d) of Ang II (b). Intracellular RS scavenging activity was measured by DCF-DA using ARBs (c). PTK activity was measured by Antibody BeaconTM tyrosine kinase assay kits (d). cSRC activation was measured using Western blot analysis to detect cSRC protein levels in aged rats. Anti-β-actin antibody was used to evaluate equal protein loading. One representative result is shown from three experiments that yielded similar results (e). Results of one-factor ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Ang II-treated group. Ang II angiotensin II, ARBs angiotensin II receptor blockers, cSrc v-src sarcoma, DCF-DA dichlorodihydrofluorescein diacetate, PTK protein tyrosine kinase, RS reactive species

Article Snippet: YPEN-1 cells were obtained from ATCC (Manassas, VA, USA).

Techniques: Activity Assay, Tyrosine Kinase Assay, Activation Assay, Western Blot

Journal: Age

Article Title: Mechanism of Ang II involvement in activation of NF-?B through phosphorylation of p65 during aging

doi: 10.1007/s11357-011-9207-7

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