Bca technique

Samples containing 30 μg of protein, quantified by BCA technique (Thermo Fisher Scientific), were applied in each lane, resolved in a 12% SDS-PAGE, and transferred to nitrocellulose membranes using standard procedures. Membranes were incubated with anti-T. cruzi trypomastigote antibodies [22], monoclonal antibody (mAb) anti-cruzipain (provided by Dr. Ana Paula Lima, Univ. Fed. Rio de Janeiro, Brazil) or mAb 39 anti-TS [23]. Binding was detected after incubation for 1 h at room temperature with the respective peroxidase-conjugated secondary antibody (goat anti-mouse or rabbit IgG) by ECL (Pierce) using an image apparatus.

Frozen lung tissue was homogenized on ice using a rotor blender (Fisher) on ice in Tissue protein extraction reagent (T-Per from Sigma). This was supplemented with phosphatase and protease inhibitors (Halt; Sigma). Homogenates were then centrifuged for 15 min at 4°C, and the supernatants were collected and frozen at −80°C until required. The protein concentration was established using a BCA technique (Thermo Scientific), and 30–40 μg of protein were then separated by electrophoresis on a Bis-Tris NuPage gel. Proteins were then transferred to PVDF Immobilon and transfer was confirmed with Ponceau red stain. The blot was blocked at room temperature for 1–2 h in 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20. Membranes were then incubated overnight at 4°C with primary antibody diluted accordingly in 5% milk/TBS-T. These were subsequently washed using TBS-T and then incubated with secondary antibody for 1–2 h at room temperature. The antibody labeling was visualized using enhanced chemiluminscence (ECL; Amersham) with exposure to autoradiographic film (GE Healthcare).

The media was discarded, and RIPA lysate was added to the cells for total protein extraction. The total protein concentration was measured using the BCA technique (Thermo, Shanghai, China). Protein samples were separated through SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% skim milk at room temperature for 1 h, the membrane was washed three times with TBST and then incubated overnight at 4 °C with the primary antibody at the recommended dilution ratio. Subsequently, the membrane was washed again with TBST and incubated at room temperature for 1 h with the DyLight 800-labeled secondary antibody (ROCKLAND, USA). The membrane underwent three additional TBST washes before imaging using a fluorescence scanning instrument (Odyssey, Danbury, CT, USA). Primary antibodies against FOXM1 (ab207298, dilution: 1/1,000), FAM83A (ab128245, dilution: 1/1,000), CDC20 (ab183479, dilution: 1/2,000), CDC6 (ab109315, dilution: 1/4,000), cyclin B (ab32053, dilution: 1/8,000), H3 (ab1791, dilution: 1/5,000), and GAPDH (ab9485, dilution: 1/2,500) were bought from Abcam for this work.