To further ascertain the nuclear translocation of Egr1, we analyzed the expression of Egr1 at a protein level in both nuclear and cytosolic fractions

To further ascertain the nuclear translocation of Egr1, we analyzed the expression of Egr1 at a protein level in both nuclear and cytosolic fractions. oxygen species (ROS)2 (19). Large quantities of ROS have been implicated as microbicidal agents in pathological situations and ultimately result in apoptosis of the macrophages harboring the pathogen, thereby resulting in parasite clearance (20). Although promastigotes are susceptible to oxygen intermediates generated employs differential induction of host SOCS proteins to subvert macrophage apoptotic machinery triggered by parasite internalization-mediated oxidative burst, thus establishing its replicative niche inside the host. EXPERIMENTAL PROCEDURES Cell Culture and Parasites The pathogenic promastigotes of strain (MHOM/IN/1983/AG83) were maintained in Medium 199 (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 50 units/ml penicillin, and 50 g/ml streptomycin. The murine macrophage cell line RAW 264.7 was maintained at 37 C, 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, penicillin (100 units/ml), and streptomycin (100 g/ml). infection experiments were carried out with the RAW 264.7 cell line using stationary phase promastigotes at a 10:1 parasite/macrophage ratio. Reagents, Antibodies, and Constructs All antibodies were from Santa Cruz Biotechnology and Cell Signaling Technology. All other chemicals were from Sigma, unless indicated otherwise. Apoptosis Detection by Annexin V Staining RAW 264.7 cells (2 106) were infected with promastigotes for different time periods. One group of infected macrophages for each time point of infection was treated with H2O2. After an hour of treatment, the culture media were replaced, and cells were incubated overnight at 37 C, 5% CO2. The cells were washed twice with PBS. Apoptosis was then determined using annexin-V-FLUOS staining kit (Roche Applied AMG 337 Science) as per the manufacturer’s instructions. Cells were analyzed on FACS Canto IITM cell sorter using 488 nm excitation and 530 nm emissions for FITC and 600 nm for PI fluorescence using FACS Diva software. Immunoprecipitation AMG 337 and Immunoblotting Cells were lysed in lysis buffer (Cell Signaling Technology), and the protein concentrations in the cleared supernatants were estimated using a protein assay (Bio-Rad). Immunoprecipitation was performed as described previously (33). Briefly, pre-cleared cell lysates (500 g) were incubated overnight with specific primary antibody at 4 C. For co-immunoprecipitation studies, pulldown with unrelated antibodies served as control. 25 l of protein A/G plus agarose beads (Santa Cruz Biotechnology) were added to the mixture and incubated for 4 h at 4 C. Immune complexes were collected and washed three times with AMG 337 ice-cold lysis buffer and once with lysis buffer without Triton X-100. The immunoprecipitated samples and cell lysates were resolved by 10% SDS-PAGE and then transferred to nitrocellulose membrane (Millipore). 30 g of protein from the whole cell lysate of each sample were loaded as input. The membranes were blocked with 5% BSA in wash buffer (TBS, 0.1% Tween 20) for 1 h at room temperature and probed with primary antibody overnight at dilution recommended by the suppliers. Membranes were washed three times with wash buffer and then incubated with alkaline phosphatase-conjugated secondary antibody and detected by hydrolysis of 5 bromo-4-chloro-3-indolylphosphate chromogenic substrate according Rabbit polyclonal to FN1 to AMG 337 the manufacturer’s instructions. Estimation of ROS Production Intracellular ROS generation was measured using the oxidant sensitive green fluorescent dye 2,7-dihydrodichlorofluorescein diacetate (H2DCFDA) (Molecular Probes). Measurement of fluorescence in cells was made by counting at least 10,000 events/test using a FACScalibur flow cytometer AMG 337 (BD Biosciences), with a fluorescein isothiocyanate filter, and the cells were gated out based on their fluorescent property. Samples were examined by FACScalibur, and the results were analyzed using CellQuest software (BD Biosciences). Phosphatase Assay Macrophages were lysed in PTP lysis buffer (50 mm Hepes, pH 7.4, containing 0.5% Triton X-100, 10% glycerol, 1 mm benzamidine,.