The reaction mixture and GSH control were analyzed in positive ion mode by electrospray ionization using a LTQ XL ion trap mass spectrometer. Real time screen shots of the chro matograms selleck chemicals llc were captured in the Xcalibur browser, ver sion 3. 3. Reduced GSH is known to produce a peak with a m z 308. QM Inhibitors,Modulators,Libraries covalently bound to gluta thione has a predicted peak at m z 413. 4 based on molecular weight calculations in Symyx Draw 3. 2. DNA ladder assay LNCaP and RWPE 1 cells were grown in 75 cm3 cell culture flasks to 80% confluence and treated with NPAA, staurosporine, or DMSO as previously described. Cells were collected Inhibitors,Modulators,Libraries as previously described and were then lysed and processed with the Apoptosis DNA Ladder Kit according to the manufacturers instructions.

The RWPE 1 and LNCaP sample DNA, the stauros porine control, and DNA ladder supplied with the kit were mixed Inhibitors,Modulators,Libraries with loading buffer and added to a 2% agarose gel containing 1 10,000 dilution of SYBR Safe. The gel was electrophoresed Inhibitors,Modulators,Libraries at 75 V for 2 hours in TBE buffer and then photographed under UV light using a ChemiDoc XRS system with Image Lab software. Protein carbonyl assay RWPE 1, LNCaP, COS 7, and COS 7 OPH cells were grown in 25 cm3 cell culture flasks to 80% confluence. The cells were then treated with NPAA or DMSO and collected as previously described. The cells were then lysed with 2% digitonin in PBS and the protein concentra tion was determined using the BCA assay kit. For each sample, an aliquot of 50 ul of protein lysate containing 5 ug ul of protein in PBS was added to two 1. 5 ml tubes. One tube was used as the negative control tube.

Inhibitors,Modulators,Libraries A volume of 200 ul of 10 selleck chem mM DNPH was added to the sample tube, and a volume of 200 ul of 2. 5 M HCl was added to the control tube. The tubes were incubated in the dark at 24 C for one hour. Proteins were precipitated by adding 500 ul of 20% TCA and incubating on ice for 5 min, followed by centrifugation at 10,000 g for 10 min at 4 C. The supernatant fluid was removed and the protein pellets were suspended in 1 ml of 1 1 ethanol ethyl acet ate followed by centrifugation at 10,000 g for 10 min at 4 C. Removal of supernatant fluid, suspension of pellets, and centrifugation were repeated three times. The super natant fluid was then removed and the protein pellets were dissolved in 300 ul of 6 M guanidine hydrochloride and mixed using a vortex mixer every 10 min for one hour. Ali quots of 200 ul from each tube were added to separate wells of a clear 96 well plate, and the absorbance at 370 nm was measured using a Spectra max plus 384 mi croplate reader. The corrected absorbance was calculated by subtracting the absorbance of the well containing the control tube aliquot from the absorbance of the well containing the sample tube aliquot.