Collection of tissue samples We routinely collected tissue samples [11–14,25,27] during the peak of the pro-life and pro-death phase (Mev group) or 30 or 180 min after microinjection of aCSF into RVLM (vehicle group). The brain was rapidly removed and placed on dry ice, and tissues from both sides of the ventrolateral medulla, at the level of RVLM (0.5 to 1.5 mm rostral to the obex) were collected by micropunches made with a stainless steel bore (1 mm i.d.) and frozen in liquid nitrogen. Medullary tissues collected from anesthetized animals but without treatment served as the sham controls. In some experi- ments, proteins from the nuclear or cytosolic fraction of the samples were extracted using a commercial kit (Active Motif, Carlsbad, CA). Total protein or that in the nuclear or cytosolic extracts was estimated by BCA Protein Assay (Pierce, Rockford, IL). Protein expression We employed Western blot analysis [11–14,25,27] to detect changes in expression of HIF-1a, HIF-1b, HIF-2a, Ubc9, NOS I, PKG, NOS II or nitrotyrosine (marker for peroxynitrite) protein, or proteins conjugated by SUMO-1. The primary antiserum used for HIF-1a, HIF-1b, HIF-2a, SUMO-1 or Ubc9 were the same as those used for immunoneutralization. The other primary antisera used included a rabbit polyclonal antiserum against NOS I (Santa Cruz, Santa Cruz, CA), NOS II (Santa Cruz), PKG (Calbiochem, San Diego, CA); or a mouse monoclonal antiserum against nitrotyrosine (Upstate Biotechnology, Lake Placid, NY) or b-actin (Chemicon, Temecula, CA). The secondary antisera used included horseradish peroxidase-conjugated donkey anti-rabbit IgG (Ge- healthcare, Uppsala, Sweden) for HIF-1a, HIF-1b, HIF-2a, SUMO-1, NOS I, NOS II, PKG; or horseradish peroxidase- conjugated sheep anti-mouse IgG (Gehealthcare) for Ubc9, nitrotyrosine or b-actin. The amount of protein was quantified by the ImageMaster software (Amersham Pharmacia Biotech, Buckinghamshire, UK), and was expressed as the ratio relative to b-actin protein. Densitometric values that were not statistically different from the background were designated below detection limits. Transcriptional activity of HIF-1 We measured HIF-1 DNA binding capacity [12] in nuclear proteins extracted from ventrolateral medulla using EMSA [54]. The 39 end of a double-stranded synthetic oligonucleotide probe for HIF-1 (59-ACCGGCCCTACGTGCT-GTCTCAC-39 and 39- TGGCCGGGATGCACGACAGAGTG-59) [54] was labeled with digoxigenin-11-ddUTP (Roche, Penzberg, Germany). DNA and protein complexes resolved on 4% polyacrylamide gels by electrophoresis were detected by chemiluminescence after reacting with an anti-digoxigenin antiserum. Competitive assay using unlabeled HIF oligonucleotide served as the negative control. A rabbit polyclonal antiserum against HIF-1a or HIF-1b (Santa Cruz) was added to the DNA binding reaction cocktail in supershift assay to determine the participation of HIF subunits. The occurrence of nucleus-bound translocation of HIF-1a was confirmed using Western blot analysis on cytosolic or nuclear proteins [11,12]. In some experiments, we quantified the transcription activity of HIF-1a using an ELISA-based HIF-1 transcription factor assay kit (Active Motif, Carlsbad, CA) [27]. In brief, HIF present in nuclear extract was allowed to bind to an oligonucleotide containing the hypoxia response element sequence (59-TACGTGCT-39). After the introduction of an antibody that recognizes an epitope on HIF-1a that is accessible on DNA binding, the addition of a secondary horseradish peroxidase- conjugated antibody provided a sensitive colorimetric readout that can be determined by spectrophotometry. HIF-1a activity was Sumoylation of HIF-1a in Experimental Brain Death quantified by measuring the absorbance at 450 nm using an ELISA microtiter plate reader (Anthros Labtec, Salzburg, Austria), and was expressed as fold changes against sham controls. Immunofluorescence staining and confocal microscopy We employed double immunofluorescence staining coupled with laser scanning confocal microscopy [11,12] to detect nucleus- bound translocation or subcellular localization of HIF-1a, HIF-1b or SUMO-1 in RVLM neurons labeled with a mouse monoclonal antiserum against a specific neuron marker, neuron-specific nuclear protein (NeuN; Chemicon). Secondary antisera (Molecu- lar Probes, Eugene, OR) used included a goat anti-rabbit IgG conjugated with Alexa Fluor 568 for HIF-1a or HIF-1b, and a goat anti-mouse IgG conjugated with Alexa Fluor 488 for NeuN; or a goat anti-rabbit IgG conjugated with Alexa Fluor 488 for SUMO-1 and a goat anti-mouse IgG conjugated with Alexa Fluor 568 for NeuN. Tissues similarly processed but omitting primary antiserum against HIF-1a, HIF-1b or SUMO-1 served as our negative controls. Immunoreactivity was viewed under a Fluor- view FV10i laser scanning confocal microscope (Olympus, Tokyo, Japan). Immunoprecipitation and immunoblot analysis We employed immunoprecipitation followed by immunoblot analysis [11,12] to establish an interaction between HIF-1b and HIF-1a in nuclear proteins, or between SUMO-1 and HIF-1a in both cytosolic and nuclear proteins extracted from ventrolateral medulla. Protein extracts were immunoprecipitated with affinity- purified goat polyclonal anti-HIF-1b, rabbit polyclonal anti- SUMO-1 or mouse monoclonal anti-HIF-1a antiserum conjugat- ed with protein G-agarose beads (Santa Cruz). Western blot analysis of HIF-1a or HIF-2a from proteins immunoprecipitated by anti-HIF-1b antiserum, HIF-1a from proteins immunoprecip- itated by anti-SUMO-1 antiserum, or SUMO-1 from proteins immunoprecipitated by anti-HIF-1a antiserum was carried out as described above. Histology In some animals that were not used for biochemical analysis, the brain stem was removed at the end of the physiological experiment and fixed in 10% formaldehyde-saline solution that contains 30% sucrose for at least 72 h. Frozen 25-mm sections of the medulla oblongata stained with neural red were used for histological verification of the microinjection sites. Statistical analysis All values are expressed as mean 6 SEM. The effects of various treatments on the averaged value of MSAP or HR calculated every 20 min after administration of test agents or vehicle, the sum total of power density for LF component in the SAP spectra over 20 min, or the protein expression level in the ventrolateral medulla during each phase of Mev intoxication, were used for statistical analysis. One-way or 2-way ANOVA with repeated measures was used, as appropriate, to assess group means. This was followed by the Scheffe´ multiple-range test for post hoc assessment of individual means. P,0.05 was considered to be statistically significant. Author Contributions Conceived and designed the experiments: SHHC AYWC. Performed the experiments: C-YT CHYW FCHL K-YD EYHS. Analyzed the data: JYHC SHHC AYWC. Wrote the paper: JYHC SHHC AYWC. Supervised the research: JYHC SHHC AYWC. PLoS ONE | www.plosone.org 12 March 2011 | Volume 6 | Issue 3 | e17375