Sumoylation of HIF-1a in Experimental Brain Death Figure 7. Activation of HIF-1 leads to upregulation of NOS I/PKG signaling in RVLM during the pro-life phase. Illustrative gels or summary of fold changes against aCSF controls in ratio of nitric oxide synthase I (NOS I), protein kinase G (PKG), NOS II or nitrotyrosine (NT; marker for peroxynitrite) relative to b-actin protein detected in ventrolateral medulla of rats that received immunoneutralization of HIF-1a, HIF-1b or HIF-2a subunit in RVLM, 30 min before induction of Mev intoxication. Note that NT is presented as % relative to b-actin because it is below detection limit (ND) in aCSF controls. Values are mean 6 SEM of triplicate analyses on samples pooled from 4–6 animals per experimental group. *P,0.05 versus aCSF group and +P,0.05 versus Mev group in the post hoc Scheff e´ multiple-range test. doi:10.1371/journal.pone.0017375.g007 the vehicle control. To avoid verbose presentation, however, the phrase ‘‘0.02% Triton X-100’’ is omitted from subsequent narration. To avoid the confounding effects of drug interactions, each animal received only one antiserum pretreatment. Evaluation of tissue hypoxia in RVLM A combined oxygen/temperature/blood flow probe (Oxford Optronix, Oxford, UK) stereotaxically positioned into RVLM was used in experiments that called for simultaneous and continuous measurement of tissue oxygen tension, temperature, and microvas- cular perfusion [15]. The dimension of the tip of the probe is approximately 800 mm. Instantaneous changes in local oxygen tension, compensated for fluctuations in tissue temperature, were processed by an OxyLite monitor (Oxford Optronix). Real-time microvascular red blood cell perfusion in tissue was processed by an OxyFlo monitor (Oxford Optronix). Laser Doppler signals from the tissue were recorded in blood perfusion units (BPU), which is a relative unit defined against a controlled motility standard. PLoS ONE | www.plosone.org 11 March 2011 | Volume 6 | Issue 3 | e17375