Sumoylation of HIF-1a in Experimental Brain Death Figure 1. Sumoylation of proteins in RVLM ameliorates failure of central cardiovascular regulation in Mev intoxication model of brain death. Temporal changes in mean systemic arterial pressure (MSAP), heart rate (HR) or power density of the low-frequency (LF) component of SAP signals in rats that received pretreatment by microinjection bilaterally into RVLM of an anti-SUMO-1 or anti-Ubc9 antiserum (1:20), normal rabbit serum (NRS,1:20; vehicle for anti-SUMO antiserum) or normal mouse serum (NMS, 1:20; vehicle for anti-Ubc9 antiserum), 30 min before local application (at arrow) of artificial cerebrospinal fluid (aCSF) or mevinphos (Mev; 10 nmol) to the bilateral RVLM. Values are mean 6 SEM, n = 5–7 animals per experimental group. *P,0.05 versus NRS+aCSF or NMS+aCSF group, and +P,0.05 versus NRS+Mev or NMS+Mev group at corresponding time-points in the post hoc Scheffe´ multiple-range test. B, baseline. doi:10.1371/journal.pone.0017375.g001 aCSF control (data not shown), which subsided during Phase II (Figure 2D). Activation of HIF-1a in RVLM also ameliorates failure of brain stem cardiovascular regulation associated with experimental brain death Our third series of experiments investigated whether HIF-1a, which is potently activated by hypoxia [22–24], also participates in brain stem cardiovascular regulation during experimental brain death. Western blot analysis (Figure 3A) on total proteins extracted from ventrolateral medulla revealed that whereas HIF-1b or HIF- 2a expression remained constant, HIF-1a level was significantly elevated during early Phase I when drastic drop in tissue oxygen level in RVLM took place. This augmented HIF-1a expression sustained throughout Phase I, and returned to baseline during Phase II Mev intoxication. HIF-1a, HIF-1b or HIF-2a expression in ventrolateral medulla of animals that received aCSF was comparable to sham controls (Figure 3A). Detailed immunoblot analysis (Figure 3B) further established a significant elevation of HIF-1a in both the nuclear and cytosolic fractions of proteins extracted from ventrolateral medulla during Phase I Mev intoxication. On the other hand, the primarily nuclear presence of HIF-1b subunit (Figure 3B) or cytosolic presence of HIF-2a (data not shown) remained constant. Again, double immunoflu- orescence staining coupled with laser scanning confocal micros- copy revealed that immunoreactivity of HIF-1a (Figure 3C) was identified primarily in the cytoplasm and HIF-1b in the nucleus of RVLM neurons in sham-control animals. During early and late Phase I, the abundant presence of HIF-1a immunoreactivity in the nucleus of RVLM neurons (Figure 3C) was accompanied by an augmented amount in the cytoplasm, which subsided during Phase II. On the other hand, there was minimal alteration in the preferential presence of HIF-1b immunoreactivity in the nucleus (Figure 3C) of RVLM neurons during both phases of Mev intoxication. PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e17375