Journal: Free radical biology & medicine

Article Title: The glutaredoxin/S-glutathionylation axis regulates interleukin 17A-induced pro-inflammatory responses in lung epithelial cells in association with S-glutathionylation of Nuclear Factor kappa B family proteins

doi: 10.1016/j.freeradbiomed.2014.04.028

Figure Lengend Snippet: (A) Mouse alveolar type II cells (C10) were stimulated with IL-17A (100ng/ml) and NF-κB was assessed in whole cell lysates by measuring phosphorylated forms of RelA (pRelA) and IκBα (pIκBα). β-actin; loading control. Right panels: densitometric evaluation of pRelA and pIκBα. Data reflect arbitrary units, following normalization to loading control, β-actin. (B) Assessment of nuclear RelA and RelB content and degradation of cytosolic IκBα following stimulation with IL-17A (50ng/ml) in C10 cells. β-actin; loading control for cytosolic lysates, (labeled as C); Histone H3 (H3) was used as a loading control for nuclear lysates, (labeled as N). Right and bottom panels: densitometric evaluation of nuclear RelA, nuclear RelB and IκBα. Data reflect arbitrary units, following normalization to respective loading controls. (C) Evaluation of mRNA expression of NF-κB-dependent negative regulators A20 and IκBα via real-time PCR in C10 cells stimulated with IL-17A (50ng/ml). Results were normalized to the housekeeping gene cyclophilin and are presented as relative expression. *p≤0.05 (ANOVA), compared to untreated controls (Sh).

Article Snippet: Antibodies The following antibodies were used in this study: rabbit anti-RelA, rabbit anti-RelB (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Histone H3, rabbit anti-IκBα (Cell Signaling Technology, Danvers, MA), mouse anti-GSH (Virogen, Watertown, MA), mouse anti-IKKα (Upstate, Millipore, Billerica, MA) Streptavidin conjugated-HRP (Jackson, West Grove, PA), goat anti-Grx1 (American Diagnostica, Stamford, CT) and mouse anti-β-actin (Sigma-Aldrich, Saint Louis, MO).

Techniques: Labeling, Expressing, Real-time Polymerase Chain Reaction

Journal: Free radical biology & medicine

Article Title: The glutaredoxin/S-glutathionylation axis regulates interleukin 17A-induced pro-inflammatory responses in lung epithelial cells in association with S-glutathionylation of Nuclear Factor kappa B family proteins

doi: 10.1016/j.freeradbiomed.2014.04.028

Figure Lengend Snippet: (A) Assessment of NF-κB activation following siRNA-mediated ablation of Grx1 or control siRNA and stimulation with IL-17A (50ng/ml). Nuclear (N) and cytosolic (C) lysates were harvested from C10 cells and nuclear content of RelA and RelB (top panels) was assessed. Histone H3 (H3); loading control. Bottom panels: Evaluation of Grx1 in cytosolic lysates to confirm siRNA-mediated ablation. β-actin; loading control. Graphs represent densitometric evaluation of nuclear RelA and RelB. Data reflect arbitrary units, following normalization to the loading Histone H3. (B) C10 cells were subjected to siRNA-mediated ablation of Grx1 or control siRNA, and stimulated with IL-17A for indicated times, and A20 mRNA expression was evaluated. Results were normalized to the housekeeping gene cyclophilin and are presented as relative expression.

Article Snippet: Antibodies The following antibodies were used in this study: rabbit anti-RelA, rabbit anti-RelB (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Histone H3, rabbit anti-IκBα (Cell Signaling Technology, Danvers, MA), mouse anti-GSH (Virogen, Watertown, MA), mouse anti-IKKα (Upstate, Millipore, Billerica, MA) Streptavidin conjugated-HRP (Jackson, West Grove, PA), goat anti-Grx1 (American Diagnostica, Stamford, CT) and mouse anti-β-actin (Sigma-Aldrich, Saint Louis, MO).

Techniques: Activation Assay, Expressing

Journal: Free radical biology & medicine

Article Title: The glutaredoxin/S-glutathionylation axis regulates interleukin 17A-induced pro-inflammatory responses in lung epithelial cells in association with S-glutathionylation of Nuclear Factor kappa B family proteins

doi: 10.1016/j.freeradbiomed.2014.04.028

Figure Lengend Snippet: (A) Assessment of nuclear RelA and RelB following siRNA-mediated ablation of IKKα or control siRNA and stimulation with IL-17A (50ng/ml). Nuclear (N) and cytosolic (C) lysates were harvested from C10 cells and nuclear content of RelA and RelB was assessed. Histone H3 (H3) was used as a loading control. Bottom panels: Assessment of IKKα in cytosolic lysates to confirm siRNA-mediated ablation. β-actin; loading control. Graphs represent densitometric evaluation of nuclear RelA and RelB. Data reflect arbitrary units, following normalization to the loading Histone H3. (B) Assessment of KC and CCL20 mRNA expression following siRNA-mediated ablation of IKKα and stimulation with IL-17A (50ng/ml) at indicated times. Values were normalized to the housekeeping gene cyclophilin, and data presented and relative expression. * p≤0.05 (ANOVA) compared to untreated controls (Sh); † p≤0.05 (ANOVA) compared to scr siRNA groups at the same time point.

Article Snippet: Antibodies The following antibodies were used in this study: rabbit anti-RelA, rabbit anti-RelB (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Histone H3, rabbit anti-IκBα (Cell Signaling Technology, Danvers, MA), mouse anti-GSH (Virogen, Watertown, MA), mouse anti-IKKα (Upstate, Millipore, Billerica, MA) Streptavidin conjugated-HRP (Jackson, West Grove, PA), goat anti-Grx1 (American Diagnostica, Stamford, CT) and mouse anti-β-actin (Sigma-Aldrich, Saint Louis, MO).

Techniques: Expressing