Int. J. Environ. Res. Public Health 2012, 9 478 groups. To quantitate As3+ effects on [3H]NEM binding to tubulin -SH groups, tubulin proteins free of MAPs prepared from the bovine brain were diluted with 10 mM phosphate buffer containing 0.3% NP40 to a final concentration of 1.5 mg/mL, pretreated with As3+ at indicated concentrations for 1 h at 0 C, then mixed with [3H]NEM (2 µCi/mL), and incubated for an additional 1 h at 37 C. Proteins were precipitated with 5% TCA and collected on nitrocellulose filters. Collected proteins on the membrane were measured by β-counting. The amount of radioactivity was normalized to total tubulin proteins and expressed as % of the control. Differences between control and As3+ treated samples (n = 3 for each group) were evaluated by using the ANOVA program as described [22]. 2.9. Cytoskeletal and Cytosolic Protein Isolation in Cultured Cells The cytoskeletal and cytosolic protein fractions in cultured cells were prepared by detergent extraction and centrifugation as described [21]. Briefly, control and treated cells were rinsed with PBS, and gently scraped into the MT stabilizing buffer (PM2G) containing 0.1 M Pipes, pH 6.9, 1 mM MgSO4, 2 M glycerol, and 2 mM EGTA, and extracted with 0.3% NP-40 in the PM2G buffer. After micro-centrifugation, the supernatant was collected and the pellet resuspended in a PM buffer (0.1 M Pipes, pH 6.9, 1 mM MgSO4) followed by centrifugation. The supernatant was mixed with the first extract and referred to as the “cytosolic” fraction. The final pellet was resuspended in 0.3% NP-40 in 10 mM phosphate buffer (pH 7.0) containing 1 mM EGTA and termed as the “cytoskeletal” fraction. Total protein concentrations in each fraction were determined by using the reagent from Bio-Rad as described [22]. 2.10. Western Blot Analysis Cytosolic and cytoskeletal lysates or total cell extracts in control and treated cells each containing equal amounts of protein (10–15 µg) were boiled in an SDS sample buffer and analyzed by SDS-PAGE gradient gel (4–15%) as described [23] using primary antibodies, i.e., the rabbit anti-tubulin (1:1,000), the mouse anti-metallothionein (1:500), the rabbit anti--GCS (1:1,000), or the rabbit anti-actin (1:1,000) antibody, and HRP conjugated corresponding secondary antibodies, i.e., the anti-rabbit or mouse IgG with 1:5,000 dilutions. Molecular weights were determined by comparison with BenchMark pre-stained protein ladder (Invitrogen). Protein bands were quantitated by the 1 D Scan EX software (Scananalytics, Fairfax, VA, USA). Experiments as shown here were repeated at least three times with reproducible results and a representative one is presented, unless otherwise indicated. 2.11. 2-D Gel Analysis Cell metabolic labeling, cytoskeletal protein extraction and 2-D gel analysis were carried out as described [25]. Briefly, growth-arrested cells were incubated with [35S]methionine at 50 µCi/mL for 24 h in methionine-free DMEM in the presence or absence of 10 M As3+. Cells treated with 5 µg/mL nocodazole, an MT depolymerization agent, at the last 3 h incubation, were used as a positive control. Cells were washed with PBS followed by PM2G with the protease inhibitor cocktail (Roche, Mannheim, Germany), extracted twice with 0.3% NP-40 in PM2G containing protease inhibitors and finally with 5 mM CaCl2 (Ca2+). Ca2+ released MT proteins including tubulins and MAPs were used