Sigma casp 3 c

Caspase-3 activity was determined with the caspase-3 Assay kit, according to the manufacturer's instructions (Sigma CASP-3-C, Sigma-Aldrich, St. Louis, Mo, USA). This assay is based on the spectrophotometric detection of the Ac-DEVD-pNA substrate after cleavage. Cells were harvested in lysis buffer [50 mm HEPES, 5 mMdithiothreitol (DTT), 5 mN CHAPS, 10 mg/mL pepstatin, benzamidine 2.5 mM, aprotinina 10 mg/mL, pepstatin 1 mg/mL, 0,5 mMphenylmethylsulfonylfluoride (PMSF), pH 7.4] and homogenized with a Teflon-glass homogenizer. Lysates were clarified by centrifugation at 10,000 Â g for 5 min, and clear lysates containing 100 mg proteins were incubated with caspase-3 substrate, at 37 C for 3 h. The concentration of the p-nitroaniline (pNA) released from the substrate is calculated from the absorbance values at 405 nm after incubating the plate at 37 C for 90 min. The activity, expressed as micromoles of p-nitroaniline per minuteper milliliter, was calculated with a p-nitroaniline calibration curve. A positive control of caspase-3 and an inhibitor-treated cell lysate control (for measuring the nonspecific hydrolysis of the substrate) were added to the plate.

Caspase-3 activity was determined with the Caspase-3 assay kit (Sigma CASP-3-C; Sigma-Aldrich), according to the manufacturer's instructions. This assay is based on the spectrophotometric detection of the Ac-DEVD-p-nitroaniline (pNA) substrate after cleavage. Cells were grown in 100 mm dishes, treated with TGF-b isoforms and harvested in lysis buffer (50 mM HEPES, 5 mM CHAPS, 5 mM DTT, pH 7.4). Lysates were clarified by centrifugation at 15,000xg for 10 min at 4 C, and clear lysates containing 200 mg proteins were incubated with caspase-3 substrate at 37 C for 3 h. The concentration of pNA released from the substrate was calculated from the absorbance values at 405 nm. The activity, expressed as picomoles of pNA per minute per milliliter, was calculated with a pNA calibration curve. A positive control of caspase-3 and an inhibitor of caspase-3 (200 mM inhibitor Acetyl-Asp-Glu-Val-Asp-al [AcDEVD-CHO]) were added to the plate.