Samples were run on a 12% acrylamide gel and stained with Coomassie brilliant blue R250 (BioRad, Hercules, CA). Excised gel slices were destained using 50% acetonitrile in 50 mM ammonium bicarbonate (pH 7.9) and vacuum dried. Samples were rehydrated with 1.5 mg/ml dithiothreitol (DTT) in 25 mM ammonium bicarbonate (pH 8.5) at 56°C for 1 h, subsequently alkylated with

10 mg/ml iodoacetamide (IAA) in 25 mM ammonium bicarbonate (pH 8.5), and stored in the dark at room temperature for 1 h. The pieces were subsequently washed with 100 mM ammonium bicarbonate (pH 8.5) for 15 min, washed twice Opaganib research buy with 50% acetonitrile in 50 mM ammonium bicarbonate (pH 8.5) for 15 min each, vacuum dried, and rehydrated with 4 μl of proteomics grade modified trypsin (100 μg/ml; Sigma, St. Louis, MO) in 25 mM ammonium bicarbonate (pH 8.5). The pieces were covered in a solution of 10 mM ammonium bicarbonate with 10% acetonitrile (pH 8.5) and incubated at 37°C for 16 h. Liquid Chromatography-Tandem Mass Spectrometry Liquid CHIR-99021 manufacturer chromatography coupled to tandem mass spectrometry (LC/MS-MS) analysis was conducted at the Mass Spectrometry Laboratory at Montana State University. Peptides were separated on a microfluidic

ChipCube interface and detected with an ESI-Trap XCT Ultra instrument (Agilent, Santa Clara, CA). The MASCOT search engine was used to compare peptide masses determined by MS to masses of sequences in the NCBInr bacterial database. Acceptable protein identifications required expectation values of 0.01 for LC-MS/MS. Microarray HFKs were grown to 90% confluence in six well plates. Cells were then treated with 2 ml BCM, PCM, or EPI for four hours. After treatment, the medium was removed and RNA was isolated using an Quisqualic acid RNeasy minikit (Qiagen, Valencia, CA) following the manufacturer’s instructions for adherent cells. Extracted RNA was ethanol precipitated and resuspended in water as previously described [71]. RNA concentrations and purity were

determined by measuring absorbencies at 260 nm and 280 nm on a GeneQuant spectrophotometer. RNA quality was also evaluated using the RNA 6000 NanoChip assay on a 2100 Bioalyzer (Agilent Technologies, Palo Alto, CA) in the Functional Genomics Core Facility at Montana State University. RNA integrity number for all samples used exceeded 9.5 on a scale to 10. Total RNA (500 ng) was reverse transcribed, amplified and biotin-labeled via in vitro transcription using the MessageAmp Premier kit (Applied Biosystems/Ambion, Austin, TX). The resulting cRNA was fragmented and hybridized to Affymetrix GeneChip Human Genome U133A 2.0 arrays (#900468, Affymetrix, Santa Clara, CA) at 45°C for 16 hours with constant rotational mixing at 60 rpm. Washing and staining of the arrays was performed using the Affymetrix GeneChip Fluidics Station 450. Arrays were scanned using an Affymetrix GeneChip Scanner 7G and GCOS software version 1.4. Microarray data were analyzed using FlexArray version 1.4.