Protein assay method

The effects of test compounds (0.1 mMe10 µM) on endogenous NQO1 activity were evaluated by the NQO1 activity assay kit (Abcam, Cambridge, MA, USA). Briefly, cell pellets were solubilized in 1× extraction buffer on ice for 20 min and then centrifuged at 18,000 × g for 20 min at 4 °C. Supernatants were transferred to new tubes and protein concentrations were determined using the BioRad protein assay method. Samples were diluted with supplemented buffer to 2 × the working concentration. Two wells were prepared for each sample (with and without inhibitor) in triplicate. The reaction buffer and the reaction buffer plus inhibitor were prepared according to the instruction of manufacturer. Absorbance was measured at 440 nm every 20 s for 5 min using the BioTek Synergy HT Multi-Mode Microplate Reader with shaking before reading.

Whole cell extracts were obtained from purified mouse B cells or cell lines treated with 1 μM PI3K inhibitors using GST-FISH buffer (10 mM MgCl₂, 150 mM NaCl, 1% NP-40, 2% Glycerol, 1 mM EDTA, 25 mM HEPES (pH 7.5)) supplemented with protease inhibitors (Roche), 1 mM phenylmethanesulfonylfluoride (PMSF), 10 mM NaF and 1 mM Na₃VO₄. Extracts were cleared by centrifugation at 12,000 r.p.m. for 15 min. The supernatants were collected and assayed for protein concentration using the Bio-Rad protein assay method. 20 μg of proteins were loaded on 12% Mini-PROTEIN TGX gels (BIO-RAD), transferred on nitrocellulose membrane (GE Healthcare), blocked with 5% Skim milk (BIO-RAD). Primary antibodies for immunoblotting included: rat monoclonal anti-mouse AID (mAID-2 clone, eBioScience, CAT NO #14-5959-82), mouse monoclonal anti-human AID (ZA001, Life Technologies, CAT NO # 39-2500), rabbit monoclonal anti-PI3K p110δ (Y387, Abcam, CAT NO #32401), rabbit polyclonal anti-β–actin (Sigma, CAT NO #A2066), rabbit monoclonal anti-Phospho-AKT (S473) (D9E, Cell Signaling Technology, CAT NO #4060), rabbit monoclonal anti-AKT (pan) (C67E7, Cell Signaling Technology, CAT NO #4691). Membranes were developed with ECL solution (GE Healthcare). AID protein abundance was measured by ImageJ software and normalized for the β-actin intensity of the corresponding lane.

Differential in-gel electrophoresis (DIGE) and mass spectroscopy protein identification studies were performed by Applied Biomics, Inc. (Hayward, CA). Pooled maternal plasma samples at gestational day 13 from BN and LEW rats (n=3 rats per strain, 100 μl plasma from each rat) were used. The plasma samples were thawed and vortexed for 20 sec. The samples were spun for 30 min at 4 °C at 14,000 rpm and the supernatant was collected. Protein concentration was measured using Bio-Rad protein assay method. For each sample, 30μg of protein was mixed with 1.0 μl of diluted CyDye, and kept in dark on ice for 30 min. BN plasma proteins were labeled with Cy3 and LEW plasma proteins with Cy5, respectively. The labeling reaction was stopped by adding 1.0 μl of 10 mM lysine to each sample, and incubating in the dark on ice for an additional 15 min. The labeled samples were then mixed together. The 2X 2-D sample buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 2% pharmalytes and trace amount of bromophenol blue), 100 μl DeStreak^™ solution and Rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/ml DTT, 1% pharmalytes and trace amount of bromophenol blue) were added to the labeling mix to make a total volume of 250 μl.