In brief, monolayers of J774A.1 cells grown in RPMI-1640 medium with 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine were withdrawn and after counting 2 × 10 cells/80 μL were added to plastic vials and incubated with 20 μL of different concentrations of pMTX-I, II, and III (1.5, 3 and 6 μg/mL) diluted in RPMI (control), for 1 h at 37°C in a humidified atmosphere (5% CO2).
20 µl aliquots of YHKB at a concentration of 0.1 mg/ml in 360 mM NaCl, 20 mM TFA, pH 1.7 were withdrawn at different aggregation times, deposited for 5 minutes on a freshly cleaved mica substrate, washed with milliQ water and dried under vacuum.
Aliquots of YHKB at a concentration of 0.1 mg/ml in 30% (v/v) TFE, 50 mM acetate buffer, pH 5.5, were withdrawn at different aggregation times and diluted 5 times with milliQ water.
For temperature stability, enzyme samples were incubated in sealed screw-cap vials, at pH 7 (phosphate buffer, 100 mM) at various temperatures, sample aliquots were withdrawn periodically, and remaining activity was determined at regular assay conditions.
cGMP generation was allowed to proceed for two minutes, after which aliquots were withdrawn and inactivated by boiling in 50 mM Tris, 4 mM EDTA buffer.
Aliquots were withdrawn over time and reactions were stopped by adding 150 mM EDTA.
Samples (1 mL) were withdrawn every 5 min, and the residual activity was measured using 2 (15 mM) as the substrate.
Aliquots were withdrawn at regular time intervals and the reaction was stopped with an equal volume of 50 mM EDTA.
Samples were withdrawn after suitable time intervals and the reaction was terminated by addition of 1 mL of 50 mM citrate buffer, pH 4.5.
His worldly honors were withdrawn.
