Trypsin inhibitor from glycine max soybean

Semi-confluent PC3 cells were rinsed twice with PBS and separated into single cells by treatment with trypsin/EDTA solution (HyClone Laboratories, Logan, UT, USA). Trypsin inhibitor from glycine max soybean (Sigma-Aldrich Co.) was added to deactivate the trypsin. The cells were pre-incubated with and without WEGST (50 µg/mL) for 30 min; 3 × 10⁵ cells were then seeded on collagen I (10 µg/mL)-coated plates and harvested at various times, as indicated. Total protein was extracted from each cell and transferred to PVDF membranes, as described above. The membranes were first used for the detection of phosphor-FAK expression using the phosphospecific antibody (p-FAK; BD Biosciences, San Jose, CA, USA), and the blot was stripped through incubation with 1 N NaOH during 1–2 min at room temperature; it was then washed three times with TBS-T and used for the detection of total FAK (t-FAK) using the mouse anti-FAK antibody (BD Biosciences, San Jose, CA, USA). The activation rate of FAK (p-FAK/t-FAK) was estimated using the Image J program.

Cultures containing at least 5% mid-trophozoite stage-infected RBCs were washed in PBS and divided into 2 equal fractions. Cells were harvested at 300 × g for 5 min and resuspended in 10 volumes of warm PBS (P) or PBS containing 1 mg/ml TPCK-Treated Trypsin (Sigma) (T). Samples were incubated at 37°C for 1 h, inverting occasionally to prevent sedimentation. Trypsin inhibitor from Glycine max (soybean) (Sigma) was added to each sample to 5 mg/ml final concentration and incubated at room temperature for 15 min. Cells were harvested and the supernatant discarded. The pellet fraction was solubilized in 10 volumes of ice cold 1% TritonX-100 (Sigma) in PBS containing cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and incubated on ice for 30 min. All subsequent steps contained 1x cOmplete EDTA-free Protease Inhibitor Cocktail (Roche). Samples were centrifuged at 16 000 × g for 10 min at 4°C, then the pellets were washed three times in the ice cold TritonX-100 solution. The pellet was solubilized in 20 volumes of 2% SDS/PBS and mixed at room temperature for 30 min. The samples were centrifuged at 16 000 × g for 10 min, the supernatant transferred to a new tube and centrifuged again. The resulting supernatant was transferred to a new tube and prepared for immunoblotting.