Journal: PLoS ONE

Article Title: Synergistic Interactions between Cytokines and AVP at the Blood-CSF Barrier Result in Increased Chemokine Production and Augmented Influx of Leukocytes after Brain Injury

doi: 10.1371/journal.pone.0079328

Figure Lengend Snippet: The activation of the JNK signaling cascade in the Z310 cell line was assessed after 1-h incubation with either TNF-α (5 ng/ml) or a combination of TNF-α (5 ng/ml) and AVP (1 nM). In non-radioactive JNK activity assays, 200 ng of recombinant (rec) human c-Jun protein was used as a substrate for JNK. The extent of activation of c-Jun and ATF2 was assessed by Western blotting (30 µg of total protein per lane was loaded). The representative immunoblots are shown based on three independent experiments. The ratios of optical density of bands for phosphorylated proteins over the optical density of bands for the total (phosphorylated and non-phosphorylated) proteins are shown.

Article Snippet: The rabbit monoclonal antibodies were as follows: anti-human c-Jun (clone 60A8; diluted 1∶200) and anti-human activating transcription factor 2 (ATF2) (clone 20F1; diluted 1∶200) both from Cell Signaling.

Techniques: Activation Assay, Incubation, Activity Assay, Recombinant, Western Blot

Journal: PLoS ONE

Article Title: Synergistic Interactions between Cytokines and AVP at the Blood-CSF Barrier Result in Increased Chemokine Production and Augmented Influx of Leukocytes after Brain Injury

doi: 10.1371/journal.pone.0079328

Figure Lengend Snippet: To inhibit the activity of JNK in rats sustaining TBI, a selective JNK inhibitor SP600125 (JNK Inh) was injected i.p. at 4 and 8 h post-TBI at a dose of 30 mg/kg for each injection. A control group of rats was injected with vehicle (Veh; DMSO). (A) The efficacy of pharmacological inhibition of JNK as assessed by the reduction in post-traumatic activation of ATF2 in the lateral ventricle choroid plexus (CP) at 6 h after injury. Another target transcription factor for JNK, c-Jun, was undetectable in the CP. The activity of ATF2 was assessed by Western blotting in both the ipsilateral (Ipsi CP/I) and contralateral (Contr CP/C) CPs (30 µg of total protein per lane was loaded). The representative immunoblots are shown based on two independent experiments involving separate groups of animals. The ratios of optical density of bands for phosphorylated ATF2 over the optical density of bands for the total (phosphorylated and non-phosphorylated) ATF2 are shown. (B) The effect of JNK inhibition on post-traumatic production of neutrophil chemoattractants CXCL1–3 by the lateral ventricle CP as assessed by real-time RT-PCR at 6 h after injury. Cyclophilin A (Cycl-A) was used for the normalization of the data. (C) The role of AVP in promoting the post-traumatic influx of neutrophils across the blood-cerebrospinal fluid barrier (BCSFB) and the effect of JNK inhibition on this influx. To define the pathophysiological role of AVP in promoting neutrophil influx across the BCSFB, AVP-deficient Brattleboro rats ( Avp di/di ) were compared with their parental Long-Evans strain (WT). The magnitude of neutrophil influx was assessed by the myeloperoxidase (MPO) activity assays at 24 h post-TBI. * p <0.05 for JNK Inh versus Veh or for Avp di/di versus WT ( n = 4–6 per group).

Article Snippet: The rabbit monoclonal antibodies were as follows: anti-human c-Jun (clone 60A8; diluted 1∶200) and anti-human activating transcription factor 2 (ATF2) (clone 20F1; diluted 1∶200) both from Cell Signaling.

Techniques: Activity Assay, Injection, Inhibition, Activation Assay, Western Blot, Quantitative RT-PCR