Tumor necrosis factor (tnf)

Each of the two experimental blocks was technically duplicated (i.e., two culture wells). Each culture well was further technically duplicated (i.e., two supernatants isolated per time point) as described. This resulted in 18 experimental samples (2 technical repeats for each of 3 different inducing cytokine concentrations at 3 different time points) and three unstimulated control samples (technical repeats).
Human recombinant IL-1β (#11457756001), IL-4 (#I4269), IL-6 (#I1395), IL-10 (#IL010), and TNF (#T6674) (all from Merck, Kenilworth, NJ) were each sourced as lyophilized powder, which was reconstituted according to the manufacturer's instructions. Serial dilution of a stock solution generated the final concentrations seen in Table 1. Each cytokine was used at three concentrations covering the range of concentrations seen in human microdialysis studies, adjusted for relative recovery determined in vivo.^{7 (link)} We also included somewhat higher doses to adjust for an early phase of injury before microdialysis monitoring was instituted (Table 1). The cytokine solution (10 μL) was added to 990 μL of culture within each well to make up the desired final concentration within 1 mL at the time of medium replacement. Cell cultures were then returned to an incubator.

Monocyte cytokine production was analyzed using freshly isolated monocytes from 8 healthy Kenyan children, 10 healthy Kenyan adults, and 10 healthy US adults, as previously described [35 (link)]. Monocytes were isolated from whole blood via negative selection with the RosetteSep Human Monocyte Enrichment Cocktail (Stemcell Technologies, 15,068). Cells were suspended in culture medium (RPMI-1640 supplemented with 2 mM L-glutamine, 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, and 0.05 mM 2-ME) and placed in 96-well polypropylene plates at 5 × 10⁴ cells per well (concentration 5 × 10⁵ cells/ml). Cells were stimulated with 10 ng/ml LPS (Sigma-Aldrich) and 100 ng/ml Pam3CSK4 (P3C) (Invivogen) and compared with a media-alone control; each condition was performed in duplicate. Cells were cultured for 18 h at 37 °C, in 5% CO₂, on an orbital shaker. Supernatants were harvested and stored at − 80 °C. A multiplex magnetic bead–based immunoassay was used to measure concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF (EMD Millipore) in the culture supernatants immediately after initial thawing.

Intravitreal injection of TNF (Sigma-Aldrich, St. Louis, MO, USA) was performed as described previously [20 (link), 21 (link)]. Briefly, rats were anesthetized with an intramuscular injection of a mixture of ketamine-xylazine (10 and 4 mg/kg, respectively). A single 2-μl injection of 10 ng of TNF in 0.01 M PBS, pH 7.40, was administered intravitreally into the right eye of an animal under a microscope to avoid lens injury. The PBS alone was administered into the contralateral left eye as a control. In the brimonidine treatment groups, 2, 20, or 200 pmol of brimonidine tartrate (Senju Pharmaceutical Co., Ltd., Osaka, JAPAN) in 0.01 M PBS was mixed with 10 ng of TNF and administered intravitreally in a simultaneous injection. A previous study used a single 5-μl intravitreal injection of brimonidine (0.85 μM to 34 μM, i.e., 4.25 pmol to 170 pmol) in rats and showed the upregulation of BDNF in RGCs [4 (link)]. Therefore, our current brimonidine dosage (2-μl intravitreal injection of 1 μM to 100 μM) is likely to be a similar concentration. The rats were euthanized 1 or 2 weeks after the intravitreal injections with an intraperitoneal overdose of sodium pentobarbital, followed by enucleation of the eyes.

The following commercial antibodies were used: HRP-conjugated anti-mouse I gG (NA931), and anti-rabbit IgG (NA941) from GE Healthcare (Pittsburgh, PA), HRP-conjugated anti-rat IgG (AP136P), and anti-ubiquitin Lys63 specific (05–1313) from EMD Millipore (Billerica, MA), anti-TAK1 (sc-7162), anti-IKKα/β.(sc-7607), anti-IκBα.(sc-371), anti-JNK (sc-7345), anti-p38 (sc-728), anti-Tab1 (sc-6052), anti-β-actin (sc-69879), and anti-ubiquitin (sc-8017) from Santa Cruz (Dallas, Texas), anti-p-TAK1 (4531), anti-p-IKKα/β.(2078), anti-p-IκBα.(9246),.anti-p-JNK (9251), and anti-p-p38 (9211) from Cell Signaling (Beverly, MA), anti-HA (11867423001) from Roche (Indianapolis, IN), and anti-Flag (M2, F3165) from Sigma (St. Louis, MO). LPS, TNF, IL-1β,.5Z-7-oxozeaenol, dithiothreitol (DTT), iodoacetamide (IAA), and formic acid were obtained from Sigma. Acetonitrile (ACN) and ammonium bicarbonate were obtained from Aldrich (Milwaukee, WI). Sequencing-grade trypsin was purchased from Promega (Madison, WI).

Wild‐type or Cuedc2^−/− MEFs were transiently transfected with 0.2 mg of the luciferase reporter pNF‐κB‐Luc, plus 0.02 μg of the Renilla reporter pRL‐TK. After being treated for 6 h with 10 ng/ml of TNF (Sigma) or hypoxia/reoxygenation, transfected cells were collected. Luciferase activity was assessed using a Dual‐Luciferase reporter Assay system (Promega). Penilla luciferase activity was used as an internal control for transfection. All experiments were repeated at least three times.