Journal: Redox Biology

Article Title: Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation ☆

doi: 10.1016/j.redox.2013.11.003

Figure Lengend Snippet: Effect of EPS on Nrf2 in SCs. Nuclear levels of active Nrf2 (A) and Nrf2 mRNA levels (B) were measured after SCs were treated with 10 or 50 µM EPS for 4 h. Values are means±SD of three experiments. *Significant difference from the value of control ( P <0.05). In C and D, SCs were transfected with control siRNA (siControl) or Nrf2 siRNA (siNrf2) and were treated or not treated with 50 µM EPS for 24 h. Subsequently, γ-GCS mRNA levels (C) and GSH levels (D) were measured. Values are means±SD of three experiments. *Significant difference from the value of siControl treated with EPS ( P <0.05).

Article Snippet: The amount of active Nrf2 in the nuclear extracts was determined by subjecting samples of 20 μg protein to assay with a TransAM Nrf2 DNA Binding ELISA Kit (Active Motif).

Techniques: Control, Transfection

Journal: Cell Death Discovery

Article Title: CYP epoxygenase metabolites of docosahexaenoic acid protect HL-1 cardiac cells against LPS-induced cytotoxicity through SIRT1

doi: 10.1038/cddiscovery.2015.54

Figure Lengend Snippet: EDPs induce mitobiogenesis in HL-1 cardiac cells. HL-1 cardiac cells were stimulated with LPS (1 μ g/ml) in the presence of 19,20-EDP (1 μ M), DHA (100 μ M) and/or MSPPOH (50 μ M) for 24 h. ( a ) Relative rates of mitobiogenesis were assessed using ELISA detecting simultaneous expression of SDH-A (nDNA-encoded protein) and COX-I (mtDNA-encoded protein) in each well of plated HL-1 cells. The ratio between COX-I and SDH-A expressions represents the relative rate of mitobiogenesis. ( b ) NRF1 DNA-binding activity was measured in the whole-cell lysates using ELISA. ( c ) NRF2 DNA-binding activity was measured in the whole-cell lysates using ELISA. ( d ) pCREB (Ser133) DNA-binding activity was measured in the whole-cell lysates using ELISA. ( e ) Whole-cell lysates were harvested and then analyzed by western immunoblotting for the levels of key transcriptional regulators of mitobiogenesis. Representative western blots and the results of quantification are demonstrated. ( f ) SIRT1 activity was measured in the whole-cell lysates by bioluminescent assay in the presence of trichostatin A (1 μ M). And the levels of NAD and NADH were determined in the cells by bioluminescent assay. Values are represented as mean±S.E.M. N =3 independent experiments. * P <0.05 treatment versus vehicle control; # P <0.05 treatment group versus LPS or LPS/MSPPOH.

Article Snippet: NRF1 DNA-binding assay was performed using an ELISA kit from Assay Biotech (Sunnyvale, CA, USA), pCREB (Ser133) DNA-binding activity was measured using an ELISA kit from Cayman Chemical, NRF2 and NF- κ B DNA-binding assays were performed using ELISA kits from Active Motif (Carlsbad, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Relative Rate, Binding Assay, Activity Assay, Western Blot