Sumoylation of HIF-1a in Experimental Brain Death Figure 6. Preferential sumoylation of HIF-1a in RVLM during the pro-life phase. (A and B) Illustrative gels or summary of fold changes against sham (S) controls of HIF-1a from proteins immunoprecipitated by anti-SUMO-1 antiserum (A); or SUMO-1 from proteins immunoprecipitated by anti-HIF-1a antiserum (B) in the nuclear or cytosolic fraction of samples collected from ventrolateral medulla of sham (S) or aCSF (V) controls during Phases MI and MII Mev intoxication. (C and D) Illustrative gels or summary of fold changes against sham (S) controls in ratio of HIF-1a relative to b-actin protein in whole cell lysate (C) or transcriptional activity of HIF-1a in nuclear extract measured by an ELISA-based HIF-1 transcription factor assay kit (D) that detects the amount of HIF-1 binding to an oligonucleotide containing the hypoxia response element sequence from rats that received pretreatment by microinjection bilaterally into RVLM of an anti-SUMO-1 or anti-Ubc9 antiserum (1:20), NRS (1:20) or NMS (1:20), 30 min before local application of aCSF or Mev (10 nmol) to the bilateral RVLM. Values are mean 6 SEM of triplicate analyses on samples pooled from 4–6 animals in each group. *P,0.05 versus aCSF, NRS or NMS group in the post hoc Scheffe´ multiple-range test. Note numbers below (A, B) or on top (C) of the gels correspond to columns in the data summary. doi:10.1371/journal.pone.0017375.g006 agents employed included Mev (kindly provided by Huikwang Corporation, Tainan, Taiwan) and aCSF that served as the vehicle control. A rabbit polyclonal antiserum against HIF-1a (Novus Biologicals, Littleton, CO), HIF-1b (Lifespan Biosciences, Seattle, WA), HIF-2a (Novus) or SUMO-1 (Cell Signaling, Danvers, MA); or a mouse polyclonal antiserum against Ubc9 (BD, San Jose, CA) was used to effect immunoneutralization. As in previous studies [11,12], 0.02% Triton X-100 (Sigma-Aldrich, St. Louis, MO) was added to facilitate transport of the antiserum across the cell membrane of RVLM neurons. Microinjection of normal rabbit serum (NRS; Sigma-Aldrich) or normal mouse serum (NMS; Sigma-Aldrich) plus 0.02% Triton X-100 served as PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17375