Next, double-distilled water was added and the cells were incubated for 4 h at 25°C to obtain total lysis. The lysates were centrifuged

at 1,400 × g for 5 min, and the supernatant underwent electrophoresis by SDS-PAGE. Proteins in the gel were blotted onto a nylon membrane; membrane strips were incubated with blocking buffer for 4 h at 25°C. Incubation for 1 h with streptavidin-HRP followed. A control containing PbMLSr was revealed with the Catalyzed Signal Amplification (CSA) System kit (DAKO). The negative control was developed with the supernatant of A549 cells after lyses (without incubation with the biotinylated protein). Confocal analysis The cellular localization of the PbMLS was performed as described by Batista et al. [55] and Lenzi et al. [56] for confocal laser scanning microscopy (CLSM). Briefly, the cells growing in different sources of carbon were fixed in 4% paraformaldehyde for 1 h, washed and centrifuged. After permeabilization with Triton find more X-100, the cells were washed in PBS and incubated in blocking solution (2.5% BSA, 1% skim milk, 8% fetal calf serum) for 20 min (Fernandes da Silva, 1988). The diluted (1:100) primary antibody anti-PbMLSr was added overnight at 4°C. After washing three times with PBS, the cells were incubated

with secondary antibody (Alexa Fluor 488 anti-rabbit Molecular Probes 1:700) for 1 hour. Before mounting in 90% glycerol in PBS, adjusted to pH 8.5, containing antifading agent (p-phenylenediamine 1 g/L) (Sigma-Aldrich), the cells were stained with Evans blue (1/10000 in 0.01 M PBS). The specimens were analyzed by laser confocal microscopy (LSM 510-META, Zeiss). Flow cytometry BV-6 in vitro assay analysis All flow cytometry analyses were performed on a BD FACSCanto (BD Biosciences) using an air-cooled argon-ion laser tuned to 488 nm and 115 mW. The flow rate was

kept at approximately 10,000 events (cells), and green fluorescence was amplified logarithmically. Ten thousand events were collected as monoparametric histograms of log fluorescence, as well as list mode data files. The data were analyzed by FACSDiva Software (BD Biosciences) and Origin Software [54]. Enzymatic activity MLS activity was determined as described by Zambuzzi-Carvalho (2009) [30]. Briefly, the enzymatic assay was carried out at room temperature. 25 mg samples were added to 500 ml assay buffer Celecoxib containing 5 mM acetyl-CoA (20 ml) and water to a volume of 980 ml. The reaction had the optical densities read at 232 nm until stabilization. The enzymatic reaction was started by the addition of 100 mM glyoxylate (20 ml). The method is based on the consumption of acetyl-CoA at 232 nm. The activity was calculated considering that one unit at 232 nm is defined as 222 nmols/mg of acetyl-CoA. The specific activities were given in U/mg protein, with U being equal at nmol/min. Statistical analysis Results are expressed as the mean ± SD of the mean of three SGC-CBP30 nmr independent experiments.