Three independent cultures as well as protein extractions

and 2D-PAGE were performed to assess the reproducibility of the experiment. Gels were stained with Coomassie Brilliant Blue (CBB). CBB staining was carried out according to Neuhoff et al. (1988) with minor modifications and scanned in a Microtek 9800XL densitometer (Microtek) at 300 dpi resolution. Gels were stored in vacuum-sealed plastic bags at 4 °C. pdquestadvance software version 8.0 (Bio-Rad) was used for spot detection and quantitation, and to assess reproducibility. Protein spots chosen for mass spectrometric analysis (MS) were excised from the gels and manually digested. The gel pieces were rinsed three times with AmBic buffer (50 mM ammonium bicarbonate in 50% Natural Product Library HPLC grade methanol (Scharlau, Spain) and once with 10 mM DTT (Sigma-Aldrich). The gel pieces were rinsed

twice with AmBic buffer and dried in a SpeedVac before alkylation with 55 mM iodoacetamide (Sigma-Aldrich) in 50 mM ammonium bicarbonate. Navitoclax mouse Once again, the gel pieces were rinsed with HPLC grade AmBic buffer (Scharlau), before being dehydrated by the addition of HPLC grade acetonitrile (Scharlau) and dried in a SpeedVac. Modified porcine trypsin (Promega) was added to the dry gel pieces at a final concentration of 20 ng μL−1 in 20 mM ammonium bicarbonate, incubating them at 37 °C for 16 h. Peptides were extracted three times by 20 min incubation in 40 μL of 60% acetonitrile in 0.5% HCOOH (formic acid). The resulting peptide extracts were pooled, concentrated in a SpeedVac and stored at −20 °C. A combination of matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF) (MS) and MALDI-TOF/TOF (MS/MS) was used for protein identification according to the following procedure. Dried samples were dissolved

in 4 μL Meloxicam of 0.5% formic acid. Equal volumes (0.5 μL) of peptide and matrix solution, consisting of 3 mg α-cyano-4-hydroxycinnamic acid (CHCA) dissolved in 1 mL of 50% acetonitrile in 0.1% trifluoroacetic acid, were deposited using the thin-layer method onto a 384 Opti-TOF MALDI plate (Applied Biosystems). Mass spectrometric data were obtained in an automated analysis loop using a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems). MS spectra were acquired in reflectron positive-ion mode with an Nd:YAG, 355-nm wavelength laser, averaging 1000 laser shots, and at least three trypsin autolysis peaks were used as internal calibration. All MS/MS spectra were performed by selecting the precursors with a relative resolution of 300 full width at half maximum and metastable suppression. Automated analysis of mass data was achieved using the 4000 Series explorer software V3.5. Peptide mass fingerprinting (PMF) and peptide fragmentation spectra data of each sample were combined through the GPSexplorer Software v3.6 using mascot software v2.1.