After washing four times with TBST, membranes were incubated with secondary goat-anti mouse alkaline phosphatase
conjugated antibody (Bio Rad Laboratories, Hercules, CA, USA; dilution 1 : 5000) during 1 h at RT. Finally, the membranes were stained using nitro blue tetrazolium and bromo-cloroindoleyl phosphate (24). Protein kinase C was purified as described previously (25). In brief, BMMϕ were homogenized in ice-cold buffer (20 mm Tris–HCl pH 7·5, 10 mm EGTA, 2 mm EDTA, 0·5% (v/v) Triton X-100, 50 mm 2-mercaptoethanol, 1 mm phenylmethylsulphonyl fluoride (PMSF), 10 μg/mL leupeptin, 0·1 mg/mL trypsin inhibitor). The suspension was frozen at −70°C during 10 min, sonicated three times during 10 min and centrifuged at 20 000 × g during 10 min. The supernatant was loaded onto DEAE-cellulose columns that had been equilibrated with column buffer (20 mm Tris–HCl pH PD-0332991 molecular weight 7·5, 50 mm 2-mercaptoethanol) this website at 4°C. After the column had been washed with column buffer, total PKC was eluted with column buffer containing 0·08 m NaCl, 2 mm EDTA and 0·1 mg/mL trypsin inhibitor. The eluate
was concentrated in an Amicon device (YM-30 membrane) (Millipore, Billerica, Massachusetts, USA) and PKCα was immunoprecipitated for the kinase assays. PKC was also purified from infected BMMϕ (5 × 106) obtained from BALB/c and C57BL/6 mice. In these cases, the BMMϕ were previously infected with 50 × 106L. mexicana promastigotes during 2 h at RT and noninfected BMMϕ were used as controls. PKCα activity was determined as described previously (26). In brief, 1 mL aliquots of partially purified and concentrated PKC (1 mg/mL) was incubated at 4°C with 1 μg/mL anti-PKCα antibody (Santa Cruz Biotechnology) for 2 h with gentle
shaking in the presence of phosphatase inhibitors (10 mmβ-glycerophosphate, 1 mm Na3VO4, 11 mm NaF, 10 mm sodium pyrophosphate and 0·2 mg/mL phosphoserine), in the absence of 2-mercaptoethanol. Then, 20 μL of Protein A-Sepharose [30% (w/v), Calbiochem, San Diego, CA, USA] were added and incubated for 2 h at 4°C. Immune complexes were then washed five times with buffer [50 mm Tris–HCl, 0·6 m NaCl, 1% (v/v) Triton aminophylline X-100, 0·5% (v/v) Octylphenyl-polyethylene glycol (IGEPAL CA-630)] containing phosphatase inhibitors and once with kinase buffer (20 mm Tris–HCl pH 7·5, 10 mm MgCl2, 0·5 mm CaCl2, 50 mm 2-mercaptoethanol). Kinase activity was analysed in immunoprecipitates incubated with the following: (i) phorbol-12-myristate-13-acetate (PMA) 1 × 10−6 m; (ii) LPG 10 μg; (iii) PMA 1 × 10−6 m combined with LPG 10 μg and (iv) Bisindolymaleimide 1 (BIM-1) 1 × 10−6 m. PKCα kinase activity was also analysed in BMMϕ obtained from L. mexicana-infected and noninfected mice of both strains.