A subset of animals undergoing I/R (n=4 in 3 hour and 6 hour reperfusion groups) received 0.6 mg/g body weight FITC dextran (4,000 kD at a concentration of 80 mg/ml; Sigma-Aldrich, St. Louis, MO) by gavage 3 hours before euthanasia. At euthanasia, plasma was harvested and the FITC concentration quantitated by fluorometry against a FITC-dextran standard curve.
Growth protocol
Animal work was approved by the Duke University and the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) Animal Care and Use Committees (ACUC) respectively. Initial studies were performed in C57BL/6 mice obtained from Jackson Lab (Bar Harbor, ME). Experiments in the previously established Ubc9 transgenic mice (Ubc9Tg) were performed in the “H3” and “N2” founder lines [25]. For mesenteric I/R, mice were anesthetized with 1% isoflurane-supplemented by a one-time administration of 80mg/kg ketamine, 5mg/kg xylazine intraperitoneally, and 0.05mg/kg buprenorphine subcutaneously. A midline laparotomy was then made and a 2-3cm ileal loop isolated and rendered ischemic by placing aneurysm clips (Kent Scientific, Torrington,CT) on the peripheral branches of the superior mesenteric artery as well as across the intestine itself to block flow through collaterals. Ischemia was maintained for 15, 30, or 45 minutes when the clamps were removed, and the incision closed. During the procedure, the body temperature was maintained at 37°C rectal temperature using a feedback-controlled heating pad. For the reperfusion phase of injury, mice were kept in a heated box with supplemental oxygen for a variable amount of time (0.5, 1, 3, or 6 hours) without anesthesia. After euthanasia, tissue was rapidly removed and snap frozen in liquid nitrogen for RNA and protein isolation or transferred into 4% paraformaldehyde (PFA) for paraffin embedding. To achieve snapshot images representative of in vivo SUMO expression and distribution, a subset of mice were euthanized, and perfused with 0.9% NaCl (10 ml) followed by 4% PFA (30 ml) via transcardiac puncture. The intestines were then removed, placed in 4% PFA overnight, transferred to PBS, and embedded in paraffin.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated using a commercially available kit (Macherey-Nagel, Bethlehem, PA), which includes a DNAse digestion step.
Label
Biotin
Label protocol
Total RNA was labeled on a sample-by-sample basis according to the manufacturer’s guidelines for use with the Mouse Clariom S GeneChip (Affymetrix, Santa Clara, CA).
Hybridization protocol
Labeled cRNA were hybridized to the Mouse Clariom S GeneChip (Affymetrix, Santa Clara, CA) in blinded interleaved fashion.
Scan protocol
The Affymetrix scanner 3000 was used in conjunction with Affymetrix GeneChip Operation Software to generate one .CEL file per hybridized cRNA.
Description
Ubc9Tg 45mins ischemia 6hrs reperfusion
Data processing
The Expression Console (Affymetrix) was used to summarize the data contained across all .CEL files and generate 28,846 SST-RMA normalized transcript cluster expression values.